RU2127602C1 - Strain "bg" of avian infection bursitis virus and vaccine against avian infection bursitis - Google Patents

Abstract

FIELD: agriculture, veterinary science, virology. SUBSTANCE: invention relates to preparing a new attenuated strain "BG" of an avian infection bursitis that can be used for specific prophylaxis and diagnosis. Strain "BG" is deposited in VGNKI collection at number "BG" "102 DEP". Vaccine against an avian infection bursitis has a viral material from the strain "BG" and a stabilizing agent at the ratio, wt.-% = 85-95/5-15. Strain "BG" is reproduced in 10-days SPF-chicken embryos at biological activity titer = 5.25-6.0 lg EID_50/0,2/ml. Fat-free milk, lactose, gelatose, sucrose and other substances are used a stabilizing agents (separately or in combination). Obtained vaccine is a homogeneous dry mass of pink-brown color in the form of water-soluble cylindric tablet. Chickens are vaccinated beginning from 10-14-days age, twice at interval for 10-14 days at the dose 100-1000 EID₅₀/ml by watering method. Immunity occurs in 14-21 days after the second vaccination and retains for total susceptible period. EFFECT: enhanced specific, immunogenic and antigenic activity and harmless of specific prophylaxis and diagnosis. 2 cl, 9 tbl, 11 ex

Description

 The invention relates to veterinary virology and biotechnology and can be used in the manufacture of agents for specific prophylaxis and diagnosis of infectious bursal disease (IBD) in birds.

 IBD of birds (Gamboro disease) is a highly contagious disease characterized by damage to the factory bag, kidneys, intramuscular hemorrhages, diarrhea. Basically, IBD affects the B-lymphocytes of a factory bag, causing atrophy of its lymphoid follicles. An important means of combating IBD, causing economic damage due to the death of 20 - 70% of birds in its acute course, reducing weight gain and the manifestation of secondary infections against the background of the immunosuppressive effect of the virus on the birds, is the timely diagnosis of the disease and its vaccination.

 IBD is acute with a characteristic clinic of the disease and subclinical. Widespread has a subclinical form. In the acute form of the disease, clinical and pathological signs characteristic of secondary infections come to the fore, which creates difficulties in making a diagnosis. Therefore, laboratory studies are crucial in making a diagnosis and require specific highly active standard antigens and sera. IBD virus strains intended for the production of antigen and immune serum should be free from contamination and have high biological and antigenic activity. The technology for the production of antigen in IBD provides for the inactivation of the virus, therefore, the strains should be sensitive to the effects of inactivating drugs and be monitored for completeness of inactivation. Existing diagnosticums are prepared on SPF chickens, SPF embryos and in cell culture.

 In foreign practice, a number of IBD virus strains are known that are used to make antigens and serums for this infection - strain "SR-1" [4], strain "1 / PY". These strains are cultured on SPF chickens (free of pathogenic factors) or in a cell culture of various origins [1]. However, the multiplication of the virus in cell culture causes a decrease in its antigenic activity. The obtained samples of antigen and serum using strains have low activity and are not amenable to standardization. In addition, the above strains are unsuitable for use as vaccines.

 It is known to use highly active virulent strains of the IBD virus for the preparation of diagnosticums.

 Known strain "cheville" used for the production of antigens and sera for laboratory diagnosis of IBD birds [1].

 However, this strain is not suitable for the manufacture of vaccines.

Also known strain "27/94" virus IBD birds to obtain diagnosticums. The strain is cultivated and titrated on commercial chickens 35 to 40 days of age and chicken embryos. The strain causes a characteristic clinical and pathological picture of the disease after 48 - 72 hours with a virus titer of 10 ^5.5 - 10 ^6.0 EID ₅₀ / ml, and the activity of blood serum reaches in the neutralization reaction 1: 1024 and in the diffusion precipitation reaction 1:64. The virus is free from contamination, specific and active [1].

 The disadvantage of this strain is its virulence, which always creates problems when observing veterinary and sanitary safety.

 Vaccine strains are unsuitable in most cases for the manufacture of diagnosticums because of their low antigenic activity.

 Known strain "52/70" virus IBD birds [2].

 The specified strain is virulent and suitable for the manufacture of only inactivated vaccines.

 Known vaccine strain "BSC" virus IBD birds, reproduced in a transplantable culture of kidney cells of the gray-green monkey VGM-70 and intended for the manufacture of vaccines [2].

 The disadvantages of this strain are its cultivation in a transplanted tissue culture, which is always a potential danger to working personnel due to the oncogenicity of the cells used, as well as its low antigenic activity.

Closest to the proposed invention in terms of essential features is the strain "VNIVIP" virus IBD birds, intended for the manufacture of a vaccine. The specified strain is an avirulent genetically homogeneous viral material that is easily cultivated in chicken fibroblast cell culture and the biological activity of the virus 10 ^5.5 - 10 ^6.0 TCD ₅₀ / ml [3].

 The disadvantage of this strain is that it has low antigenic activity.

 The problem of creating effective vaccine preventative measures for IBD in Russia has become very acute with the advent of a number of foci of the disease in the period from 1991 to 1995. The use of domestic and imported biological products in foci of infection did not provide the expected effect. This is because their use was not preceded by a study of the antigenic properties of circulating virus isolates, specific antibodies were not determined in birds before and after vaccination, as well as the level of passive maternal immunity in chickens.

 Known vaccine against IBD birds based on the strain "52/70" [2].

 The disadvantage of this vaccine is that the strain "52/70" is virulent, therefore, it is suitable for the manufacture of only inactivated vaccines.

 Also known is a vaccine against IBD in birds based on the BCF strain adapted to the transplanted culture of the kidney cells of the gray-green monkey VGM-70 [2].

 The disadvantages of this vaccine are that for reproduction of the virus they use the transplantable gray-green monkey cell line, which has not yet been adequately studied with respect to the oncogenic effects on the personnel involved in the preparation of the drug, and the effects of the poultry meat immunized with this vaccine on the human body .

Closest to the proposed invention in terms of essential features is a vaccine against IBD in birds, including avirulent genetically homogeneous viral material from the strain "VNIVIP" and a stabilizer taken in equal proportions. The specified strain is reproduced in a culture of chicken fibroblast cells with a biological activity of 10 ^5.5 - 10 ^6.0 TCD ₅₀ / ml. Skim milk is used as a stabilizer. For the prevention of IBD, the bird is immunized up to 60 days of age by feeding a vaccine with water at a dose of 10 ^3.0 - 10 ^4.0 TCD ₅₀ / ml [3].

 The disadvantage of this vaccine is that it has low immunogenic activity.

 The task of creating the present inventions was to obtain a strain of IBD virus of birds with high biological, immunogenic and antigenic activity in their native form and after inactivation and suitable for the production of both diagnosticums and live and inactivated vaccines, as well as the development of a live and inactivated vaccine based on it possessing high immunogenic activity and harmlessness.

 The technical result from the use of the proposed invention is to increase the specific antigenic and immunogenic activity and harmlessness of the means of specific prophylaxis and diagnosis.

 The specified technical result is achieved by creating a group of inventions that form a single inventive concept. The group includes inventions, one of which is intended for use in another invention.

 The specified technical result was achieved by creating an attenuated strain "BG" virus IBD birds. Strain "BG" is a new, previously unknown. The original virus for obtaining the strain "BG" was isolated from the factory bags of sick broilers of the Uralskaya poultry farm in the Orenburg region in 1993. The production strain "BG" was obtained by repeated consecutive passages on 10-day-old SPF chicken embryos.

 The strain "BG" was deposited in the State Virus Collection of the All-Russian State Research Institute for Control, Standardization and Certification of Veterinary Medicines (VGNKI) 06/21/96 under the registration number "BG" 102 DEP.

 Compared with the prototype, the strain "BG" is harmless and has a higher biological, antigenic and immunogenic activity in its native form and after inactivation. It has been experimentally confirmed that it can be used for the preparation of live (Table A, 6, 7, 8, 9,), inactivated (Table 5) vaccines and diagnostic preparations (Table 1, 2, 3, 4).

 The strain "BG" virus IBD birds characterized by the following features and properties.

 Morphological properties.

 The “BG” strain of the IBD virus of birds belongs to the Birnaviridae family, the genus Avibirnavirus, serotype I, has morphological characteristics characteristic of the IBD causative agent: the form of the virion is ecosahedral, the size of the virion is 58-60 nm. Capsid - T13, no shell.

 Antigenic properties.

By its antigenic properties, the strain "BG" refers to serovariant 1. The virus is stably neutralized by homologous antiserum. The virus does not exhibit hemagglutinating activity. During vaccination, the virus induces the formation of specific antibodies detected in RDP at a dilution of 1: 4-1: 64, in ELISA - 1: 400-1: 10000 and more, with hyperimmunization - in RDP up to 1: 4096 and higher, in ELISA> 1 : 10000. When determining the antigenic correspondence of the vaccine strain "BG" with field isolates of the IBD virus, we compared the variable region of VP ₂ 37 field isolates isolated in 1993 - 1996 in the Russian Federation and the CIS countries and 7 samples of commercial live vaccines. For this, PCR was amplified using specific oligonucleotide primers and thermostable Tag DNA polymerase, the variable region of the VP ₂ gene encoding the main conformational etitope. Using the direct sequencing method, the nucleotide sequence of this most important in the manifestation of antigenic properties of the genome of the field isolates and the vaccine strain IBD was determined. It was found that most of the field isolators of the IBD virus isolated at Russian poultry farms have antigen differences from imported vaccine strains of Ron-Merier (France), Intervet (Holland), Solvey Dufar (USA), Safit (Israel) ) by 5 - 8% and have a structure close to the vaccine strain "BG".

 Biotechnological characteristics.

 The strain "BG" exhibits high biological, immunogenic and antigenic activity both in its native form and after inactivation, as well as a high degree of homology (98%) compared with the large group of isolates (30) isolated on the territory of the Russian Federation in 1993-1996 .

Strain "BG" is intended for use as a raw material for the preparation of live and inactivated vaccines and diagnostic biologics. The strain "BG" is reproduced in 10-day SPF chicken embryos at 37.7 ^o C and a humidity of 55 - 64%. During 72 - 96 hours of incubation, the virus accumulates up to 5.25 - 6.0 lg EID _{50 / 0.2} ml. The strain "BG" is stable and harmless.

 Chemo- and genotoxonomic characteristics.

Strain "BG" is an RNA-containing virus with mol. wt. 2.16 • 10 ⁶ D. Nucleic acid is a 2-strand, 2-segment (A and B). The virus has a protein coat containing 5 types of proteins (VP ₁ - VP ₅ ). Lipids are absent. The protein shell detects the following enzymes:
VP ₁ - RNA-dependent RNA polymerase;
VP ₂ and VP ₃ are structural proteins;
VP ₄ - protease;
VP ₅ - not identified.

Strain "BG" exhibits genetic stability. The bulk of the mutations is concentrated in VP ₂ . Amino acid changes in VP ₂ determine antigenic variability.

Nucleic acid segment A contains 2 partially overlapping open reading frames for small VP ₅ and large polyprotein precursor proteins VP ₂ and VP ₃ . Segment B encodes VP ₁ .

 Physical properties

The mass of the virion is 55 • 10 ⁶ D. The sedimentation coefficient is 460S in the sucrose gradient. The floating density is 1.33 - 1.34 g / ml in a CsCl gradient.

 Resistance to external factors.

 The strain "BG" is resistant to solvents (ether, chloroform) and detergents, sensitive to formaldehyde, UV radiation, y-radiation and drying.

 Additional features and properties.

 Immunogenic activity - 100%.

 Reactogenicity is absent.

 Pathogenicity is absent.

 Virulence is absent.

 Oncogenicity is absent.

 There is no contagiousness.

 The attenuation stability was observed during 5 passages on sensitive chickens.

 According to the applicant, the proposed strain meets the conditions of patentability "novelty" and inventive step ".

The specified technical result was also achieved by creating a vaccine against IBD birds, characterized by the following set of features:
1) a vaccine against IBD birds;
2) virus-containing material from the attenuated strain of "BG" of the IBD virus of birds reproduced on 10-day-old SPF embryos of chickens with a biological activity titer of 5.25 - 6.0 lg EID _{50 / 0.2} ml, at a concentration of 85 - 95 wt. %;
3) the stabilizer is 5 to 15 wt.%.

 As a stabilizer, skim milk, lactose, gelatose, sucrose, etc. can be used separately or in combination.

The present invention includes a combination of essential features that provide a technical result in all cases for which legal protection is sought:
1) a vaccine against IBD birds;
2) virus-containing material from the attenuated strain of "BG" of the IBD virus of birds reproduced on 10-day-old SPF embryos of chickens with a biological activity titer of 5.25 - 6.0 lg EID _{50 / 0.2} , at a concentration of 85 - 95 wt.% ;
3) the stabilizer is 5 to 15 wt.%.

Compared with the prototype, an essential distinguishing feature of the invention is the use of the attenuated strain "BG" of the IBD virus as a virus-containing material reproduced in 10-day-old SPF chicken embryos and with a biological activity titer of 5.25 - 65.0 lg EID _{50 / 0.2} ml

 Compared with the prototype virus vaccine based on the attenuated strain of "BG" has a higher immunogenic activity, is harmless and is able to overcome maternal antibodies against IBD.

 The analysis of the prior art by the applicant, including a search by patents and scientific and technical sources of information, and the identification of sources containing information about analogues of the invention, allowed to establish that the applicant did not find a source characterized by features that are identical (identical) to all the essential features of the claimed invention. The definition from the list of identified analogues of the prototype, as the closest in the totality of the features of the analogue, made it possible to establish a set of significant distinctive features in relation to the applicant’s perceived technical result in the proposed vaccine set forth in the independent claim.

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

 To verify the conformity of the proposed vaccine with the condition of patentability "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 vaccine does not follow for the specialist explicitly from the prior art set forth in the corresponding section of the description (no solutions having features matching the distinctive 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 vaccine meets the condition of patentability "inventive step".

 The essence of the proposed invention is illustrated by examples of their implementation.

 Example 1. Preparation of diagnostic antigen.

 SPF chickens of 14-21 days of age are inoculated with the strain "BG" (introacular, intranasal or oral). After 72 chickens they kill, take bursa, freeze. Then the bursa is finely ground, triturated with sterile glass sand, mixed with 1: 1 phosphate buffer, frozen twice, centrifuged for 1 min at 3000 rpm, the supernatant is drained, inactivated and used for work.

 The results of studies to determine the activity of the IBD antigen in the RDP are presented in table. 1.

 Example 2. Preparation of diagnostic serum.

 SPF chickens of 14 - 21 days of age are inoculated with an orally live vaccine from the "BG" strain of the IBD virus. After 14 days, an inactivated antigen is administered intramuscularly, using oil as an adjuvant, and after two weeks the chickens are bled and receive the gene immune active serum and determine its activity in RDP and ELISA. The research results are presented in table. 2.

 Example 3. preparation of diagnostic serum.

 SPF chickens of 14 to 21 days of age are given an orally live vaccine from the "BG" strain of the IBD virus. After 14 days, an inactivated antigen is administered intramuscularly using alumina hydrate as an adjuvant. After 2 weeks, the chickens are bled, receive serum and determine its activity in the RDP and ELISA.

 The research results are presented in table. 3.

 Example 4. Preparation of diagnostic serum.

 SPF chickens of 14 to 21 days of age are given an orally live vaccine from the "BG" strain of the IBD virus. After 14 days, the inactivated antigen is inoculated intramuscularly, oil is used as an adjuvant, after 10 days the inactivated antigen is reintroduced and after 7 days the chickens are bled, serum is obtained and its activity is determined.

 Stabilizing additives are added to the tested serum and it is lyophilized. Lyophilized serum is used for ELISA and RDP kits. The results of the study of the obtained serum are presented in table. 4.

 Example 5. Preparation and testing of inactivated vaccines.

 An inactivated vaccine against IBD in birds is prepared from the strain "BG" of the virus grown in developing SPF chicken embryos. The virus is inactivated with aziridine, adsorbed onto aluminum hydroxide with the addition of saponin.

 The results of studies to determine the immunogenic activity of inactivated vaccines from strain "BG" are presented in table. 5.

 Example 6. Preparation of attenuated vaccine.

At the first stage, matte material is prepared from the strain "BG" of the IBD virus of birds. The resulting virus-containing liquid is used for mass charging of 10-day-old SPF chicken embryos. Viral material in an amount of 0.2 ml is inoculated on chorioallantoic membranes (CAO) and incubated at 37.7 ^° C for 48 - 120 hours. Fallen embryos are cut, frozen at -40 ^° C, and the resulting raw material is used to prepare the vaccine. The virus-containing material is thawed, crushed in a colloid mill, extracted at 4 ^° C for 45 minutes, frozen twice - thawed, pH 7.5 phosphate buffer is added and centrifuged for 20 minutes at 3000 rpm. The virus-containing supernatant is carefully drained under sterile conditions, and then a stabilizer is added, which can be used (separately or in combination) skim milk, lactose, gelatose, sucrose, etc., packaged in vials, frozen at -60 - (- 80 ^o C) and lyophilized. The lyophilized material is corked under vacuum and tested for immunogenicity (in 14-day-old SPF chickens) and for harmlessness (in 30-day-old SPF chickens).

 The resulting vaccine is a dry, homogeneous mass of pink-brown in the form of a cylindrical tablet, soluble in water. When dissolved in water to its original volume, it turns into a stable homogeneous suspension of pink-brown color.

The vaccine is used in farms where clinical and pathological signs of the manifestation of IBD are recorded. Chicks are vaccinated from 10 to 14 days of age twice with an interval of 10 to 14 days at a dose of 100 to 1000 EID ₅₀ / ml by the method of feeding.

 Immunity in birds occurs 14 to 21 days after the second vaccination and lasts throughout the entire period susceptible to IBD.

 The resulting vaccine against IBD birds has the optimal component composition in wt.% (See table. A).

The resulting vaccine, when titrated on 10-day-old SPF embryos of chickens, has a titer of 5.25 - 6.0 lg EID _{50 / 0.2} ml.

 Example 7. Test dry attenuated vaccine.

Tests of a vaccine containing, wt.%:
Virus-containing material from attenuated strain "BG" of the IBD virus of birds reproduced on 10-day-old SPF embryos of chickens with a biological activity titer of 5.25 - 6.0 lg EID _{50 / 0.2} ml - 85
Stabilizer - 15
The immunogenic activity of the vaccine was determined in chickens. For this, 10-fold dilutions of the vaccine were preliminarily prepared and 10 chickens were immunized with each dilution by feeding. The immunogenic activity of the lyophilized vaccine was evaluated according to the results of the control infection of chickens in the experimental and control groups 14 days after administration of the vaccine. The experiments were carried out three times. The research results are given in table. 6.

 The data table. 6 indicate that a vaccine diluted 1000 times provides 100% protection of chickens, drinking a vaccine with water is an effective way of mass vaccination of birds.

 Example 8. Test dry attenuated vaccine.

Tests of a vaccine containing, wt.%:
Virus-containing material from attenuated strain "BG" of the IBD virus of birds reproduced on 10-day-old SPF embryos of chickens with a biological activity titer of 5.25 - 6.0 lg EID _{50 / 0.2} ml - 90
Stabilizer - 10
The determination of immunogenic activity was carried out on chickens. For this, 10-fold dilutions of the vaccine were preliminarily prepared, and 10 dilution of chickens were immunized by each dilution. The immunogenic activity of the lyophilized vaccine was evaluated according to the results of the control infection of chickens in the experimental and control groups 14 days after administration of the vaccine. The experiments were carried out three times. The research results are given in table. 7.

 The data table. 7 indicate that a vaccine diluted 1000 times provides 100% protection for chickens, and drinking the vaccine with water is an effective way of mass vaccination of birds.

 Example 9. Test dry attenuated vaccine.

Tests of a vaccine containing, wt.%:
Virus-containing material from the attenuated strain of “BG” of the IBD virus of birds reproduced on 10-day-old SPF embryos of chickens with a biological activity titer of 5.25 - 6.0 lg EID _{50 / 0.2} ml - 95
Stabilizer - 5
The determination of immunogenic activity was carried out on chickens. For this, 1-fold dilutions of the vaccine were preliminarily prepared and 10 dilution of chickens were immunized by each dilution. The immunogenic activity of the lyophilized vaccine was evaluated according to the results of the control infection of chickens in the experimental and control groups 14 days after administration of the vaccine. The experiments were carried out three times. The research results are given in table. eight.

 The data table. 8 indicate that a vaccine diluted 1000 times provides 100% protection for chickens, and drinking the vaccine with water is an effective way of mass vaccination of birds.

 Example 10. Comparative evaluation of virus vaccines against IBD birds.

 In the experiments, vaccines were used from the Taviar strain (Italy), from the D-78 strain (Holland), from the Gumboral ST strain (France), from the VNIVIP strain (Russia) and from the BG strain ( Russia).

 A study of the effectiveness of these vaccines was carried out on SPF chickens of 14 days of age.

 All chickens were orally inoculated with one dose of one of the above vaccines. Evaluation of the effectiveness of vaccines was carried out according to the results of the control of infection with a virulent strain 52/70 IBD of all groups of chickens 14 days after their vaccination. The research results are given in table. nine.

The data table. 9 indicate that during the control infection of vaccinated chickens with a virulent strain 52/70 IBD at a dose of 10 ⁴ TCD ₅₀ / ml 100% protection is provided only by a vaccine prepared from strain "BG".

 Example 11. Test dry attenuated vaccine.

 In laboratory experiments and in the conditions of poultry farms of the Russian Federation, a comparative assessment of the immunogenicity of various domestic and imported live vaccines against IBD was carried out and it was shown that dry virus vaccine against IBD from the “BG” strain is a more reliable and effective biological product compared to imported vaccines.

Thus, the above information indicates the fulfillment of the following set of conditions when using the proposed inventions:
- strain "BG" virus IBD birds and a vaccine against IBD birds, embodying the invention, are intended for use in agriculture, namely in veterinary medicine and biotechnology;
- for the proposed inventions in the form as described in the independent claims, the possibility of their implementation using the means and methods described in the application or known prior to the priority date is confirmed;
- strain "BG" virus IBD birds and a vaccine based on it, obtained in accordance with the proposed invention, have a high specific, immunogenic and antigenic activity, harmlessness and areactogenicity.

 Therefore, the proposed group of inventions meets the condition of patentability "industrial applicability".

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Claims (2)

 1. The strain of the virus of infectious bursal disease of birds Bursitis infectiosa gallinal cem. Birnaviridal, pog. Avibirnavirus, serotype VGNKI N "BG" 102-DEPT, for the production of specific prophylaxis and diagnosis of infectious bursal disease of birds. 2. Vaccine against infectious bursal disease of birds, containing virus-containing material from an attenuated strain of the virus of infectious bursal disease of birds and a stabilizer, characterized in that as a virus-containing material it contains an attenuated strain of the virus of infectious bursal disease of birds VGNKI N "BG" 102-DEPT reproduced on 10-day-old SPF embryos of chickens with a biological activity titer of 5.25 - 6.0 lg EID _{50 / 0.2} ml in the following ratio of components, wt.%:
Virus-containing material from an attenuated strain of the virus of infectious bursal disease of birds VGNKI N "BG" 102-DEPT reproduced on 10-day-old SPF embryos of chickens with a biological activity titer of 5.25 - 6.0 lg EID _{50 / 0.2} ml - 85 - 95
Stabilizer - 5 - 15

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