RU2233671C1 - Inactivated vaccine against egg yield reducing syndrome-76 in poultry - Google Patents

Abstract

FIELD: veterinary virology, biotechnology, vaccines.

SUBSTANCE: vaccine comprises antigenic material from the strain "BISS № 113" of egg yield reducing syndrome-76 in poultry reproduced in 9-11 day old duck embryos or SPF-chicken embryos, aminoethylethyleneinine as an inactivating agent and oily adjuvant taken in the effective ratio. The strain is deposited in the Collection of microorganisms VGNKI at registration name "BISS № 113 - DEP". The strain "BISS № 113" is reproduced in duck embryos or SFP-chicken embryos with biological activity at least 6.0 lg EID 50/0.2 cm³ and elicits RGA-heme-agglutinating activity at least = 1:4096. Prepared vaccine represents homogenous emulsion of white color. Vaccine promotes to formation of active immunity against egg yield reducing syndrome-76 in grafted poultry in 14-21 day after a single administration of preparation with the period effect for 12 months.

EFFECT: valuable properties of vaccine.

12 cl, 6 tbl, 7 ex

Description

The invention relates to the field of veterinary virology and biotechnology and can be used in the development and manufacture of vaccines for the prevention of egg-laying syndrome-76 (SSW-76) birds.

SSYA-76 is a viral disease of laying hens, characterized by a sharp, but short-term decrease in egg productivity, the appearance of depigmented eggs, eggs with a weak shell and no shell, as well as an increase in the percentage of fighting and notching.

The disease was first described in 1976 in Holland. Later, SSN-76 was recorded in many other countries in Europe, South America, Australia and Africa. In the early eighties of the last century, cases of SSN-76 were detected in our country, which was confirmed by the detection of anti-hemagglutinins to the virus SSJ-76 in the blood of sick birds.

The causative agent of the disease is a DNA-containing virus belonging to the Adenoviridae family, which, unlike representatives of other avian adenoviruses, is able to agglutinate chicken erythrocytes. The laying hens of all breeds are susceptible to the disease during the period of the highest egg production (180-240 days), but bird damage is possible in any period of the productive cycle. Chickens of highly productive meat-and-egg and meat crosses are more susceptible to the disease.

According to many foreign and domestic researchers, the economic damage caused by SSN-76 consists of a loss of 5 to 30 eggs per laying hen, and in breeding herds, taking into account culling, can reach up to 50 eggs.

Damage from the disease is exacerbated by the costs of recreational activities.

Currently, the main method for eliminating SNF-76 birds is the specific prevention of disease with inactivated vaccines (1).

However, the problems of increasing their antigenic activity, safety and stability continue to be relevant and are the main areas of research to improve these drugs.

Inactivated vaccine against BWW-76 of birds is known, containing antigenic material from CNCM strain No. 1043 of a homologous virus reproduced in a sensitive biological system, an inactivant and an oil adjuvant in an effective ratio (2, 3).

An inactivated vaccine against BWW-76 is known, containing antigenic material from strain "B 8/78" of a homologous virus reproduced in a sensitive biological system, bis-ethyleneiminoethyl oxamide as an inactivant, and an oil adjuvant in an effective ratio (4).

A known inactivated vaccine against BWW-76 birds containing antigenic material from the strain VGNKI No. 98 "B-93" of a homologous virus reproduced in a sensitive biological system, an ethyleneimine dimer as an inactivant and an oil adjuvant in an effective ratio (5).

An inactivated vaccine against BWW-76 of birds is known, containing antigenic material from the VNIVIP strain of a homologous virus enclosed in a liposomal vesicle coated with a mixture of sugars and water-soluble polymers in a ratio of 5-15: 1 (6).

The closest to the invention according to the set of essential features is an inactivated vaccine against SSW-76 birds containing antigenic material from strain "8/78" homologous virus reproduced in 9 - 11-day-old duck embryos (UE), theotropin as an inactant and oil adjuvant in the effective ratio (7).

Significant disadvantages of the known vaccine preparations, including the prototype vaccine, are their low antigenic and hemagglutinating activity (GA activity) due to the instability of the virus strains used for their manufacture during inactivation, as well as the need to cultivate the virus only on developing UEs.

The objective of the present invention was to develop an inactivated vaccine against BWN-76 birds based on a new production strain of a homologous virus with high antigenic and HA activity, retaining its native immunobiological properties during inactivation and suitable for the manufacture of a highly effective inactivated vaccine for which antigen can be obtained both in developing duck and chicken embryos (CE).

The technical result from the use of the present invention is to increase antigenic and GA activity and stability of the drug and expand the arsenal of means of specific prophylaxis of SSW-76 birds, providing intense and long-lasting immunity in the vaccinated bird.

The specified technical result was achieved by the creation of an inactivated vaccine against SSW-76 birds, characterized by the following set of features:

1. Inactivated vaccine against SSW-76 birds.

2. Antigenic material from an infectious agent strain reproduced in a sensitive biological system in an effective amount.

3. As the causative agent of the infection, the strain “BISS No. 113” of the virus CCN-76 birds in an effective amount.

4. Antigenic material from the strain “BISS No. 113” of the BSS-76 virus of birds, reproduced in 9 - 11-day UE.

5. Antigenic material from the strain “BISS No. 113” of the BSS-76 virus of birds, reproduced in 9 - 11-day UEs, with biological activity of at least 6.0 Ig EID 50 / 0.2 cm ³ .

6. Antigenic material from the strain “BISS No. 113” of the BSS-76 virus of birds, reproduced in 9 - 11-day UE, with GA activity of at least 1: 4096.

7. Antigenic material from the strain “BISS No. 113” of the BSS-76 virus of birds, reproduced in 9 - 11-day-old embryos of SPF-hens.

8. Antigenic material from the strain “BISS No. 113” of the BSS-76 virus of birds, reproduced in 9 to 11-day-old embryos of SPF-chickens, with biological activity of at least 6.0 Ig EID 50 / 0.2 cm ³ .

9. Antigenic material from the strain “BISS No. 113” of the BSS-76 virus of birds, reproduced in 9 - 11-day-old embryos of SPF-chickens, with HA activity of at least 1: 4096.

10. Inactivant.

11. Of the inactivants of aminoethylethyleneimine (AEEI).

12. AEEI in the form of a 10% aqueous solution.

13. AEEI at a concentration of 0.2-0.4%.

14. Oil adjuvant.

15. Of the oil adjuvants, the oil adjuvant is Montanide IZA-70.

16. Antigenic material from the strain “BISS No. 113” of the BSS-76 virus of birds, AEI inactivant and oil adjuvant in the ratio, wt.%:

Antigenic material 30.0-40.0

AEEI (10% aqueous solution) 0.2-0.4

Oil adjuvant Up to 100.0

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

1. Inactivated vaccine against SSW-76 birds.

2. Antigenic material from an infectious agent strain reproduced in a sensitive biological system.

3. As the causative agent of the infection, the strain “BISS No. 113” of the virus CCN-76 birds in an effective amount.

4. Inactivant.

5. Oil adjuvant.

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

1. Antigenic material from the strain “BISS No. 113” of the BSS-76 virus of birds, reproduced in 9 - 11-day UE.

2. Antigenic material from the strain “BISS No. 113” of the BSS-76 virus of birds, reproduced in 9 - 11-day UEs, with biological activity of at least 6.0 Ig EID 50 / 0.2 cm ³

3. Antigenic material from the strain “BISS No. 113” of the BSS-76 virus of birds, reproduced in 9 - 11-day UE, with GA activity of at least 1: 4096.

4. Antigenic material from the strain “BISS No. 113” of the BSS-76 virus of birds, reproduced in 9 - 11-day-old embryos of SPF-hens.

5. Antigenic material from the strain “BISS No. 113” of the BSS-76 virus of birds, reproduced in 9 - 11-day-old embryos of SPF-chickens, with biological activity of at least 6.0 Ig EID 50 / 0.2 cm ³ .

6. Antigenic material from the strain “BISS No. 113” of the BSS-76 virus of birds, reproduced in 9 - 11-day-old embryos of SP-chickens, with GA activity of at least 1: 4096.

7. Of inactivants AEI.

8. AEEI in the form of a 10% aqueous solution.

9. AEEI at a concentration of 0.2-0.4%.

10. Of the oil adjuvants, the oil adjuvant “Montanide IZA-70”.

11. Antigenic material from the strain “BISS No. 113” of the BSS-76 virus of birds, AEI inactivant and oil adjuvant in the ratio, wt.%:

Antigenic material 30.0-40.0

AEEI (10% aqueous solution) 0.2-0.4

Oil adjuvant Up to 100.0

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

1. Inactivated vaccine against SSW-76 birds.

2. Antigenic material from an infectious agent reproduced in a sensitive biological system.

3. Inactivant.

4. Oil adjuvant.

Compared with the prototype vaccine, the proposed vaccine has significant distinctive features, namely, it contains antigenic material from the strain "BISS No. 113" virus SSYA-76 birds 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. Antigenic material from the strain “BISS No. 113” of the BSS-76 virus of birds, reproduced in 9 - 11-day UE.

2. Antigenic material from the strain “BISS No. 113” of the BSS-76 virus of birds, reproduced in 9 - 11-day UEs, with biological activity of at least 6.0 Ig EID 50 / 0.2 cm ³

3. Antigenic material from the strain “BISS No. 113” of the BSS-76 virus of birds, reproduced in 9 - 11-day UE, with GA activity of at least 1: 4096.

4. Antigenic material from the strain “BISS No. 113” of the BSS-76 virus of birds, reproduced in 9 - 11-day-old embryos of SPF-hens.

5. Antigenic material from the strain “BISS No. 113” of the BSS-76 virus of birds, reproduced in 9 - 11-day-old embryos of SPF-chickens, with biological activity of at least 6.0 Ig EID 50 / 0.2 cm ³ .

6. Antigenic material from the strain “BISS No. 113” of the BSS-76 virus of birds, reproduced in 9 - 11-day-old embryos of SPF-chickens, with GA activity of at least 1: 4096.

7. Of inactivants AEI.

8. AEEI in the form of a 10% aqueous solution.

9. AEEI at a concentration of 0.2-0.4%.

10. Of the oil adjuvants, the oil adjuvant “Montanide IZA-70”.

11. Antigenic material from the strain “BISS No. 113” of the virus SSA-76 birds, AEI inactivant and oil adjuvant in the ratio, wt.%:

Antigenic material 30.0-40.0

AEEI (10% aqueous solution) 0.2-0.4

Oil adjuvant Up to 100.0

The proposed vaccine has a higher antigenic and GA activity and stability compared to the prototype vaccine and expands the arsenal of means of specific prophylaxis of SSF-76 birds.

The achievement of the technical result from the use of the proposed vaccine is explained by the fact that antigenic material from the new strain “BISS No. 113” of the BSS-76 virus of birds, which has high biological, antigenic and GA activity and stability, capable of reproducing as in developing ducks, was introduced into its composition. and in chicken embryos and providing a vaccine that creates a tense and lasting immunity in a vaccinated bird.

An additional technical result from the use of the invention is achieved due to the fact that the vaccine contains AEI as an inactivant, which can significantly reduce labor and energy costs for the manufacture of the vaccine and improve the quality of antigenic material. An additional technical result from the use of the present invention in order to increase the antigenic and GA activity of the target product is also achieved through the use of the Montanide IZA-70 oil adjuvant in its composition.

The initial virus for obtaining the strain “BISS No. 113” was isolated from organs of the reproductive system of chicken with signs of acute form of SSN-76 in 1999 in the Ulyanovsk region. The production strain “BISS No. 113” of the SSJ-76 virus was obtained by conducting 4 consecutive passages on developing UEs.

The resulting strain was deposited in the All-Russian state collection of microorganism strains used in veterinary medicine and livestock, the All-Russian Scientific Research Institute for Control, Standardization and Certification of Veterinary Medicines (VGNKI) of the Ministry of Agriculture of the Russian Federation on October 5, 2000 under the registration name "BISS No. 113-DEPT". The strain "BISS No. 113" of the SSJ-76 virus has high biological and antigenic activity in the native form and after inactivation. The possibility of its use for the preparation of an inactivated vaccine against BWN-76 birds, which creates effective protection of chickens against the specified pathogen, has been experimentally confirmed.

The strain "BISS No. 113" virus SSA-76 birds is characterized by the following features and properties.

Morphological properties

The strain "BISS No. 113" of the SSJ-76 virus belongs to the family Adenoviridae, genus Aviadenovirus, to the 3rd serological group. During electron microscopic examination, the strain "BISS No. 113" is represented by icosahedral virions that are devoid of a supercapsid membrane and do not contain lipids. The diameter of the nucleocapsid is 70 ± 8 nm.

Antigenic properties

By its antigenic properties, the strain “BISS No. 113” belongs to the 3rd serological group of avian adenoviruses. The virus is stably neutralized by homologous antiserum. In the cross-reaction of inhibition of hemagglutination (RTGA) with specific sera to strains “B 8/78”, VGNKI No. 98 “B-93” and “BISS No. 113” of virus SSYA-76 and with heterologous serums to paramyxovirus-1 (PMV-1 ), causing Newcastle disease, and orthomyxoviruses of 5-7 serotypes, the “BISS No. 113” strain was identified as a virus similar to the reference “B 8/78” and the well-known domestic VGNKI No. 98 “B-93” strains of the SSN-76 virus, and assigned to adenoviruses of birds of 3 serological groups.

14-21 days after infection of 120-day-old chickens with the virus of the strain “BISS No. 113”, the appearance of virus-neutralizing and GA antibodies is noted. The GA titer of the strain reaches 18.0-21.0 log ₂ . Samples of extraembryonic fluid (EEG) after the strain had been reproductive for UE were studied in a hemagglutination reaction (RGA). The studied samples of EEG had GA activity at a dilution of 1: 32768-1: 262144.

Biotechnological characteristics

Strain "BISS No. 113" is intended for the manufacture of an inactivated vaccine against SSW-76 birds. The strain multiplies well in 9 - 11-day UE infected in the allantoic cavity. The infectious activity of the strain after 120 hours at a temperature of 37.0 ± 0.5 ° C reaches 6.75 ± 0.31 Ig EID ₅₀ / 0.2 cm ³ .

The strain is also cultivated in CE and the primary cell culture of duck fibroblasts, causing a cytopathic effect (CPP). The results of the cultivation of the virus SSN-76 strain "BISS No. 113" are presented in table. 1. The strain is genetically stable and retains its properties for 10 passages.

Hemagglutinating properties

Strain "BISS No. 113" has pronounced hemagglutinating properties. Red blood cells of chickens and ducks agglutinate, causing the formation of a medium-grained RGA with a limit titer of 21.0 log ₂ .

Infectious properties

Chickens, ducks and geese with intratracheal, oral and intramuscular infections are susceptible to the strain BISS No. 113. Starting from the 14th day after the introduction of the virus to 120-day-old chickens, the appearance of depigmented eggshells depigmented is noted. Within four weeks, the percentage of eggshells and soft shells is about 30% or more.

The virus does not cause clinical signs of the disease in experimental and natural infections. Pathological changes are not expressed and are determined only histologically by changes in the reproductive organs.

Chemogenotaxonomic characteristic

The core of the virion contains a double-stranded DNA molecule and two internal proteins. The nucleic acid mass reaches 17.3% of the mass of the virion.

The bulk of the proteins of the virus SSV-76 birds is concentrated in the capsid shell of the virion. Core proteins account for up to 20% of all virus proteins. Core protein "1" with a molecular weight of 46 × 10 ³ g / mol is easily separated from the internal nucleotide containing DNA in complex with arginine-rich core protein "2".

Hexon, peptone base and its filament (fiber) of the BISS virus No. 113 are constructed from polypeptides with molecular weights of 120 × 10 ³ , 70 × 10 ³ , 62 × 10 ³ g / mol, respectively.

Protein structures of the virus strain "BISS No. 113" form several antigens. In the structure of the structural viral antigen, type-specific, group-specific, subgroup-specific antigens of peptone filament were found in them. The structure of the structural viral antigen revealed three main components, designated A, B and C. Antigen A is structurally associated with hexon, antigen B with peptone, antigen C with strands of virion (fiber). Hexon-linked component A contains a group-specific antigen (a) and a type-specific antigen (ε). The antigenic factor associated with peptone is called the β antigen, and with the filament of the virion (fiber) - the γ antigen.

Using PCR and sequencing of amplified genomic patches, it was found that the allantoic fluid from UE infected with the BISS strain No. 113 contained the genome of the SSN-76 virus.

The nucleotide sequence of the amplified conserved fragment of the hexon gene of strain “BISS No. 113” had complete homology with reference strains of the virus SSN-76.

Physical properties

The mass of the virion is 252 × 10 ³ g / mol. Floating density 1.32-1.35 g / ml in a gradient of CsCl. The sedimentation constant of the mature virion is 8 ^-10 × 10 ^-13 - 9 ^-10 × 10 ^-13 s.

Resistance to external factors

The strain "BISS No. 113" is stable at pH in the range of 6.0-9.0. Resistant to fat solvents. Inactivated at 56 ° C.

Additional features and properties

Antigenic activity - 100%.

Pathogenicity is expressed.

Virulence is expressed.

Contagiousness is expressed.

Oncogenicity is absent.

Free from contamination of bacterial and fungal flora, mycoplasmas and hemagglutinating viruses.

Based on the data obtained, it can be argued that the strain "BISS No. 113" in terms of antigenic and immunological spectra is an original, taxonomically new, previously unknown isolate of the virus of BSS-76 birds.

The analysis of the prior art by the applicant, including a search by patent and scientific and technical sources of information and identification of sources containing information about analogues of the invention, allowed to establish 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 aggregate of essential features of the analogue made it possible to establish a set of essential distinctive features of the proposed vaccine relative to the applicant's technical result, as set forth in the independent claim.

Therefore, the claimed invention meets the condition of patentability “novelty”.

To verify the conformity of the proposed solution to the patentability condition “inventive step”, an additional search for 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 effects of the transformations provided for by the essential features of the proposed invention have not been identified to achieve a technical result.

Therefore, the proposed vaccine meets the condition of patentability “inventive step”.

The essence of the invention is illustrated by examples of its implementation.

Example 1

The initial virus for obtaining the strain "BISS No. 113" was isolated from the organs of the reproductive system of chicken with signs of acute form of SSN-76 in 1999 in the Ulyanovsk region. The vaccine strain was obtained at the All-Russian Research Institute for the Protection of Animals by conducting sequential passages on developing UEs of 9–11 days old. To obtain the first passage virus, infected embryos were incubated in a thermostat for 120 hours at a temperature of 37.0 ± 0.5 ° C. The death of embryos during the first 24 hours after the introduction of the virus was considered non-specific. After 120 hours of incubation, all embryos were cooled at 4-8 ° C for 12-18 hours and opened. EEGs were taken from the embryo cavity with a pipette and collected in glass vials. The virus-containing fluid was subjected to low-speed centrifugation to free it from coarse birds. The clarified suspension was used for subsequent passage.

The resulting virus was subjected to comprehensive control in accordance with the OIE Guidelines for Standard Diagnostic Methods and Vaccines (1996) and, at passage level 4, was stored as a mother seed. The virus of seed brood with a titer of infectious activity of 6.75 ± 0.31 Ig EID ₅₀ / 0.2 cm ³ , presented as a virus-containing EEG, is stored at a temperature of minus 40 ° C.

The resulting strain of virus SSJA-76 birds assigned the copyright name "BISS".

Example 2

For the manufacture of an inactivated emulsion vaccine, the production strain BISS No. 113 of the SSN-76 virus obtained from VGNKI is used. The specified strain is used to obtain matrovogo seed. For this, 3 ampoules of lyophilized virus are dissolved in 30 cm ^{3 of a} sterile 0.9% sodium chloride solution containing 200 U / cm ^{3 of} streptomycin.

Cooked virus infect 150 UE or embryos of SPF-chickens 9-11 days old by introducing it into the allantoic cavity of 0.2 cm ³ . Infected embryos are incubated at a temperature of 37.4 ± 0.5 ° C and relative humidity of 60-70% for 96-120 hours. Every 24 hours, the embryos are ovoscopically. In 96-120 hours after infection and incubation, the embryos are used to obtain the virus. Before opening, the embryos are cooled at 4-8 ° C for 12-24 hours. After that, a virus-containing liquid is taken from them through a puncture in the chorioallantoic membrane with a sterile pipette. The collected liquid is centrifuged at 1000 rpm for 15 minutes and poured into 50-100 cm ³ in sterile bottles with a capacity of 100-200 cm ³ . In sterile conditions, 3-5 cm ^{3 of} liquid is taken from each vial to control sterility and HA activity. The resulting mother seed should be sterile and have a GA activity of at least 1: 4096. Frosted brood is stored frozen at a temperature not exceeding minus 40 ° C.

The mother seed is used to produce a production virus virus. To do this, the vials with the mother virus are thawed in a water bath at 18-20 ° C, the contents of the vials are purified with chloroform and centrifuged at 2500 rpm for 20 minutes. The supernatant is collected in sterile vials. The activity of the virus is determined in the RGA and the volume is adjusted by diluting with a 0.9% sodium chloride solution to an activity of 2048-4096 GAE / 0.2 cm ³ . The mother virus thus prepared is used to produce a production virus virus.

The resulting virus infects a batch of UE or embryos of SPF-chickens in an amount that ensures the production of the planned volume of the vaccine series. The infected embryos are incubated at a temperature of 37.0 ± 0.5 ° C and relative humidity 60-70% for 96-120 hours. 96-120 hours after infection, the embryos are cooled at 4-8 ° C for 12-24 hours, open the infected embryos and collect virus-containing material. The resulting virus-containing material is subjected to primary purification by centrifugation. Centrifugation is carried out for 7-10 minutes at 1000 rpm and a temperature of 2-6 ° C. At the same time, the allantoic fluid is freed from shell fragments, cells and red blood cells. The purified virus-containing EEG is poured into glass 20 dm ³ sterile bottles. The obtained EEG is subjected to inactivation by aminoethylethyleneimine (AEEI). For this, the virus-containing EEG is heated in a water bath to 37.0 ± 0.5 ° C and a pH of 7.4-7.8. With stirring, a 10% AEEI solution is added to the suspension to a final concentration of 0.2-0.4%. Inactivation is carried out for 48 hours after adding the inactivant at 37.0 ± 0.5 ° C with periodic stirring. At the end of inactivation, the bottles with the obtained antigen are cooled at 4-8 ° C for 2 days, periodically mixing the suspension. Then from each bottle a sample is taken in an amount of 20 cm ³ to determine the titer in the RGA, control, avirulence, sterility and pH. The inactivated antigen must be sterile and avirulent, have a GA titer of at least 1: 8192 and a pH in the range of 7.4-7.8. The antigen is stored at 4-8 ° C. The resulting antigen is subjected to additional purification from ballast impurities in a flow ultracentrifuge and a filter unit with an EPRS element. After that, the antigenic material is combined with an oil adjuvant in a ratio of 3: 7-2: 3 by weight. Before the preparation of the vaccine, the volume of inactivated antigen is adjusted according to the hemagglutinin titer. The GA activity of the inactivated antigen in the RGA should be at least 1: 4096. For the manufacture of emulsion vaccines, both domestic and foreign oil adjuvants can be used. In this case, an oil adjuvant of Seppic (France) Montanid IZA-70 is used.

The manufacture of inactivated emulsion vaccines is as follows. In the reactor with a sterile oil adjuvant with the stirrer turned on (250-300 rpm), the required volume of inactivated material is supplied at a speed of 2-3 dm ³ / min (pre-emulsion stage). The pre-emulsion is then passed through the homogenizer for 3 hours, resulting in a stable water-oil emulsion. Given the thermolability of the viral antigen, the emulsification process is carried out at a temperature of 4-8 ° C. Upon receipt of the emulsion control some of its characteristics: the type of emulsion, stability, viscosity and other properties. The finished vaccine is poured into 200-450 cm ³ vials, which are hermetically sealed and rolled up with aluminum caps. The resulting vaccine is controlled by the following indicators: in appearance and color, the presence of impurities and defects in the vials, the stability of the emulsion, the determination of relative viscosity, density of inactivation of the vaccine and its antigenic activity.

In appearance, the drug is a homogeneous white emulsion. During storage, peeling of oil is allowed in the upper part of the bottle. With shaking, the uniformity of the emulsion is restored.

The resulting vaccine against SSW-76 birds has the optimal component composition:

Antigenic material 30.0-40.0

AEEI (10% aqueous solution) 0.2-0.4

Oil adjuvant Up to 100.0

The antigen content in the proposed vaccine in the above range is its effective amount in the drug, ensuring the achievement of a technical result.

The proposed vaccine is intended for the prophylactic immunization of young chickens against SSN-76. Vaccination is subject to a clinically healthy bird aged 90-120 days, but no later than one month before the start of oviposition. The vaccine at a dose of 0.5 cm ³ is administered to the bird once subcutaneously in the middle third of the neck, intramuscularly in the chest area or in the fat fold of the tail. The vaccine induces immunity to infection in at least 80% of vaccinated chickens, inducing the accumulation in the blood serum of antibodies to the SSJ-76 virus in titer 5 log ₂ and above in RTGA or more than two minimum values in ELISA. The duration of post-vaccination immunity is at least 12 months.

Example 3

Studies were carried out to study the antigenic activity of the proposed vaccine against BWW-76 birds, made as described in example 2, and containing, wt.%:

Antigenic material

from the strain “BISS No. 113”,

reproduced in UE

9 to 11 days old

with biological activity

6.5 Ig EID 50 / 0.2 cm ³ and

GA activity 1: 4096 30.0

AEEI (10% aqueous solution) 0.2

Oil adjuvant Up to 100.0

Three prototypes of the preparation were made on the basis of the Montanid IZA-70 oil adjuvant. Inactivated SSN-76 virus was diluted 1: 2 with sterile PBS before vaccine formulation. As a control, we used a sample of a serial inactivated vaccine against SSN-76 prepared from strain B 8/78. All vaccines were administered to the chickens of the experimental and control groups (20 animals in each group). The results of studies to determine antigenic activity are presented in table. 2.

From the data table. Figure 2 shows that all three samples of the inactivated vaccine against SSN-76 prepared from the BISS strain No. 113, induced the formation of antibodies to the SSN-76 virus in high titers in vaccinated chickens. The vaccine from the strain "B 8/78" caused the formation of antibodies to the SSN-76 virus in titers of the same level in immunized chickens.

Example 4

Studies have been conducted to study the tension and duration of post-vaccination immunity created by an inactivated vaccine against BWW-76 birds, made as described in example 2, and containing, wt%:

Antigenic material

from the strain “BISS No. 113”,

reproduced in UE

9 to 11 days old

with biological activity

6.5 Ig EID 50 / 0.2 cm ³ and

GA activity 1: 4096 40.0

AEEI (10% aqueous solution) 0.4

Oil adjuvant Up to 100.0

Beginning from 30 to 120 days after vaccination of the bird with the vaccine received, with a frequency of 1 time per month, the intensity of humoral immunity in RTGA was evaluated. The results are presented in table. 3.

According to the table. Figure 3 shows that, in the range from 1 to 6 months, the vaccinated bird of both groups had antibodies to the SNF-76 virus in high titers. Significant differences in the level of post-vaccination immunity caused by vaccines from the strains "BISS No. 113" and "8/78" have not been established. The data obtained allowed us to conclude that a single vaccination of chickens with an inactivated preparation from the BISS strain No. 113 at a dose of 0.5 cm ³ ensured the formation of a strained level of antibodies to the SSJ-76 virus throughout the study period.

Example 5

A comparative assessment of the antigenic activity of an inactivated vaccine based on the strain “B 8/78” and an inactivated vaccine against BWW-76 birds made on the basis of the strain BISS No. 113 as described in example 2, and containing, wt.%:

Antigenic material

from the strain “BISS No. 113”,

reproduced in UE

9 to 11 days old

with biological activity

6.5 Ig EID 50 / 0.2 cm ³ and

GA activity 1: 4096 30.0

AEEI (10% aqueous solution) 0.2

Oil adjuvant Up to 100.0

Vaccines were prepared on the basis of the Montanid IZA-70 oil adjuvant in the ratio of adjuvant to antigen 7: 3 by weight. The antigen was previously diluted with the FBI in a ratio of 1: 3.

The activity of whole antigen in the RGA was as follows:

strain "BISS" - 21 log ₂ ;

strain "8/78" (sample No. 9) - 19 log ₂ ;

strain "B 8/78" (sample No. 11) - 19 log ₂ ;

strain "8/78" (sample No. 16) - 19 log ₂ ;

strain "8/78" (sample No. 17) - 19 log ₂ .

The vaccines were emulsified for 3 minutes at 3000 rpm.

Vaccines were tested for antigenic activity, vaccinating birds at the age of 100 days intramuscularly at a dose of 0.5 cm ³ , and when checking the safety of vaccines, they were administered at a dose of 1.5 cm ³ and the birds were kept for 10-12 days. All vaccines did not cause any abnormalities in the bird, both in behavior and at the injection sites. Blood serum was taken after 7, 11, 30, 60 and 90 days after vaccination and examined in rtga, and the results were expressed in log ₂ . The research results are given in table. 4.

According to the table. 4 it follows that the vaccine from the strain "BISS No. 113" and samples No. 9 and 17 of the vaccine from the strain "B 8/78" have the highest antigenic activity.

Example 6

Studies were carried out to determine the dynamics of the formation of post-vaccination antibodies after the application of an inactivated emulsion vaccine against BWW-76 birds, made as described in example 2, and containing, wt.%:

Antigenic material

from the strain “BISS No. 113”,

reproduced in UE

9 to 11 days old

with biological activity

6.5 Ig EID 50 / 0.2 cm ³ and

GA activity 1: 4096 30.0

AEEI (10% aqueous solution) 0.4

Oil adjuvant Up to 100.0

The research results are given in table. 5.

Example 7

Studies were conducted to evaluate the antigenic activity of an inactivated vaccine against BWW-76 birds, made as described in example 2, and containing, wt.%:

Antigenic material

from the strain “BISS No. 113”,

reproduced in CE

9 to 11 days old

with biological activity

6.5 Ig EID 50 / 0.2 cm ³ and

GA activity 1: 4096 30.0

AEEI (10% aqueous solution) 0.2

Oil adjuvant Up to 100.0

The research results are given in table. 6.

Thus, the above information indicates the fulfillment when using the invention of the following set of conditions:

- inactivated vaccine against BWW-76 birds, embodying the invention, is intended for use in agriculture, namely in veterinary virology and biotechnology;

- for the proposed invention in the form 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 vaccine against BWW-76 birds, a technical result was achieved consisting in increasing the antigenic and GA activity and stability of the drug and expanding the arsenal of means of specific prophylaxis of BWW-76 birds, providing intense and prolonged immunity in the vaccinated bird.

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

Sources of information

1. Syurin V.N. and other viral diseases of animals. - M.: VNITIBP, 1998, p. 846-870.

2. Pat. France No. 2382500, C 12 K 5/00, A 61 K 39/32, 09/29/1978

3. Baxendale W., Lutticken D. et al. The results of field trials conducted with an inactivated vaccine against the Egg Drop Syndrome-76 (EDS-76). Avian Pathology, 1980, v. 9, No. 1, p. 77-91.

4. Pat. RF No. 1809836, C 12 N 7/00, A 61 K 39/00, 04/15/1993

5. Pat. RF №2097066, А 61 К 39/12, С12 N 7/00, А 61 К 39/235, 11/27/1997

6. Pat. RF No. 2155030, A 61 K 9/127, 39/00, 39/235, 08.27.2000

7. Pat. RF №2183968, A 61 K 39/12, 06/26/2002 (prototype).

Claims (12)

1. Inactivated vaccine against the egg-laying syndrome-76 (SSS-76) of birds, containing antigenic material from an infectious agent reproduced in a sensitive biological system, an inactivant and an oil adjuvant, characterized in that it contains antigenic material from a strain of virus SSS-76 of birds, collection of VGNKI “BISS No. 113-DEP”, in an effective amount. 2. The inactivated vaccine according to claim 1, characterized in that it contains antigenic material from the strain "BISS No. 113-DEPT" virus CCYA-76 birds, reproduced in 9-11-day-old duck embryos (UE). 3. The inactivated vaccine according to claim 1 or 2, characterized in that it contains antigenic material from the strain "BISS No. 113 - DEPT" virus SSA-76 birds with biological activity of at least 6.0 Ig EID 50/0, 2 cm ³ . 4. The inactivated vaccine according to any one of claims 1 to 3, characterized in that it contains antigenic material from the strain "BISS No. 113 - DEPT" virus SSYA-76 birds with hemagglutinating activity of at least 1: 4096. 5. The inactivated vaccine according to claim 1, characterized in that it contains antigenic material from the strain "BISS No. 113 - DEPT" virus SSYA-76 birds, reproduced in 9-11-day-old embryos SPF - chickens. 6. The inactivated vaccine according to claim 1 or 5, characterized in that it contains antigenic material from the strain "BISS No. 113 - DEPT" virus CCYA-76 birds with biological activity of at least 6.0 Ig EID 50/0, 2 cm ³ . 7. Inactivated vaccine according to any one of claims 1, 5 and 6, characterized in that it contains antigenic material from the strain "BISS No. 113 - DEPT" virus SSYA-76 birds with hemagglutinating activity of at least 1: 4096. 8. The inactivated vaccine according to claim 1, characterized in that among the inactivants it contains aminoethylethyleneimine (AEEI). 9. The inactivated vaccine of claim 8, characterized in that it contains AEEI in the form of a 10% aqueous solution. 10. Inactivated vaccine according to any one of paragraphs.8 and 9, characterized in that it contains AEEI in a concentration of 0.2-0.4%. 11. The inactivated vaccine according to claim 1, characterized in that of the oil adjuvants it contains the oil adjuvant “Montanide IZA-70”. 12. Inactivated vaccine according to any one of claims 1 to 11, characterized in that it contains antigenic material from the strain “BISS No. 113 - DEPT” of virus CCYA-76 birds, AEEI and oil adjuvant in the ratio, wt.%: Antigenic material 30.0-40.0 AEEI 0.2-0.4 Oil adjuvant Up to 100.0