RU2268936C1 - Strain "k-58" of avian bursal infectious disease for preparing diagnostic and vaccine preparations - Google Patents

Abstract

FIELD: veterinary virology, biotechnology.

SUBSTANCE: invention relates to the strain "K-58" that is deposited in the Collection FGU VGNKI at the registration number '"K-58" № 122 - DEP". The strain is reproduced in 9-11-old SPF-chicken embryos with titer value of biological activity 5.5-7.0 lg EID₅₀/0.2 cm³. The strain is stable and harmless. Its attenuation stability is found in carrying out 5 passages in sensitive chickens. The strain shows high biological, immunogenic and antigenic activities in native state and after inactivation and useful for preparing highly specific and sensitive diagnostics and harmless live and inactivated vaccine preparations.

EFFECT: valuable veterinary properties of strain.

7 tbl, 8 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 - (infectious bursitis, Gamboro disease) is a widespread highly contagious viral disease of chickens, mainly aged 15-80 days, characterized by damage to the factory bag, kidneys, intramuscular hemorrhages and diarrhea.

IBD is acute, with a characteristic clinic of the disease, and subclinically. Widespread has a subclinical form. In the acute form of the disease, clinical and pathological anatomical signs characteristic of secondary infections come to the fore, which creates difficulty in making a diagnosis. Therefore, laboratory studies are crucial in making a diagnosis and require specific highly active standard antigens and sera.

The strains of the IBD virus of birds used for the production of antigen and immune serum must be sufficiently virulent, free from contamination and have high biological and antigenic activity. The technology for the production of antigen in IBD involves the inactivation of the virus, therefore, the strains should be sensitive to the effects of generally recognized inactivating drugs and be monitored for completeness of inactivation.

IBD birds registered in all countries of the world. In recent years, IBB has become widespread in most large poultry farms in Russia.

The economic damage from IBD caused by the death of chickens up to 20-70% in the acute course of the disease, significant culling of birds, decreased productivity, and also as a result of secondary infections, is very large.

Currently, it is generally recognized that the only correct and reliable directions in the fight against this infection are timely diagnosis of the disease and its vaccination. For specific prevention of IBD, live and inactivated vaccines are used.

A well-known feature of the causative agent of IBD is the antigenic variability of strains within the same serotype, occurring at different time intervals and in different territories and depending on the species and breed composition of the bird population, its immune status and many other factors.

In this regard, the choice of the vaccine strain of the IBD virus of birds is carried out taking into account the antigenic structure of the epizootic isolate, and for the preparation of vaccine preparations, production strains are used that have the highest degree of homology in the field of antigenic determinants with epizootic isolates of the IBD virus (1).

In foreign practice, a number of strains of the IBD virus of birds known for the manufacture of diagnostic preparations for this infection are known: strain "SR-1" (4) and strain "1 / PY". These strains are reproduced on SPF chickens (free of pathogenic factors) or in cell culture of various origins (2).

However, the multiplication of the virus in cell culture causes a decrease in its antigenic activity. Samples of antigen and serum obtained from these strains have low antigenic 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 of birds for the manufacture of diagnosticums.

The known strain "cheville" used for the production of antigens and sera for laboratory diagnosis of IBD birds (2). However, this strain is not suitable for the manufacture of vaccines.

Also known is a strain of STB virus No. 2105 of the IBD virus for birds to obtain diagnosticums. The strain is reproduced and titrated on commercial chickens 35-40 days old and chicken embryos. The strain causes a characteristic clinical and pathoanatomical picture of the disease after 48-72 hours with a virus titer of 5.5-6.0 lg EID ₅₀ / cm ³ , the activity of blood serum reaches in the neutralization reaction (pH) 1: 1024 and in the diffusion precipitation (RDP) reaction ) 1:64. The virus is free from contamination, specific and active (2).

The disadvantage of this strain is its virulence, which always creates problems when observing the requirements of the veterinary-sanitary regime.

In most cases, vaccine strains are unsuitable for the manufacture of diagnosticums because of their low antigenic activity.

The known Winterfield-2512 strain of the IBD virus of birds reproduced on 9-11-day-old embryos of SPF-chickens and used to prepare vaccines (3, 4, 5).

A known strain BIA 52/70 of the IBD virus for the preparation of vaccines is reproduced in the fabric of factory bags of chickens of 30-40 days of age or on the allantoic membranes of chicken embryos (6, 7, 8).

The known strain D-78 (ATCC No. VR-2041) of the IBD virus of birds, reproduced on 9-11-day-old embryos of SPF-hens and used for the preparation of vaccines (9).

The known strain GP / 82 (NCAJM No. V (P) -001033) of the IBD virus of birds, reproduced in cell culture of fibroblasts of embryos of SPF chickens and used to prepare vaccines (10).

The known strain "VNIVIP" virus IBD birds used for the manufacture of vaccines (11).

A known “BSC" strain of the IBC virus of birds, reproduced in the transplanted culture of kidney green-monkey monkey kidney cells VG M-70 and used for the preparation of vaccines (12).

The known Winterfield 84 - DEP strain of bird IBD virus is reproduced in chicken embryo fibroblast cell culture or on 9-11-day-old SPF chicken embryos and used for the manufacture of vaccines (13).

The known strain "Bio-92 No. 115 - DEPT" of the IBD virus of birds reproduced in the culture of chicken embryo cells or in the culture of quail embryo cells and used for the manufacture of diagnostic and vaccine preparations (14).

Common disadvantages of the known strains are their low antigenic and immunogenic activity.

Closest to the proposed invention in terms of essential features is the strain "BG" No. 102 - DEPT "of the IBD virus of birds reproduced on 9-11-day-old embryos of SPF-chickens and used for the manufacture of diagnostic and vaccine preparations (15).

By its antigenic and immunogenic activity, the strain “BG” No. 102 - DEP is superior to all known strains of the IBD virus of birds, from which vaccines were prepared for use in the Russian Federation and the CIS countries (16).

However, the prototype strain has the disadvantage that it slightly induces bursa reduction.

Thus, the problem of searching, selecting and studying epizootic isolates of the IBD pathogen to obtain new production strains of the IBD virus of birds that have high biological, antigenic and immunogenic activity both in their native form and after inactivation and suitable for the manufacture of highly specific and sensitive diagnostics and highly immunogenic and harmless vaccine preparations remains relevant.

The task of creating the present invention was to obtain a new production strain of the IBD virus of birds, which has high biological, immunogenic and antigenic activity in its native form and after inactivation and is suitable for the manufacture of diagnostic tools and specific prophylaxis of IBD in birds.

The technical result from the use of the present invention is to expand the arsenal of IBD virus strains of birds that have high biological, immunogenic and antigenic activity in their native form and after inactivation and are suitable for the manufacture of highly specific and sensitive diagnostics and highly immunogenic and harmless live and inactivated vaccine preparations.

The specified technical result was achieved by obtaining an attenuated strain "K-58" (copyright) virus IBD birds. The strain is new, previously unknown.

The original virus for obtaining the strain "K-58" was isolated from the factory bags of sick broilers at the Vasilievskaya poultry farm in the Penza region in 1994. The production strain "K-58" was obtained by repeated consecutive passages of the original isolate on 9-11-day-old embryos of SPF hens and SPF chickens.

The strain "K-58" was deposited in the All-Russian state collection of strains of microorganisms used in veterinary medicine and animal husbandry, Federal State Institution "All-Russian State Center for the Quality and Standardization of Medicines for Animals and Feed" (FGU VGNKI) Ministry of Agriculture of the Russian Federation on June 1, 2004 under registration number - strain "K-58 No. 122 - DEPT" virus IBD birds.

Compared with the prototype strain, the strain "K-58 No. 122 - DEPT" is harmless and has a higher biological, antigenic and immunogenic activity in its native form and after inactivation. The possibility of its use for the preparation of diagnostic and vaccine preparations has been experimentally confirmed.

The strain "K-58 No. 122 - DEPT" virus IBD birds characterized by the following features and properties.

Morphological properties

The strain "K-58 No. 122 - DEPT" of the IBD virus belongs to the Birnaviridae family, the genus Avibirnavirus, serotype I and has morphological characteristics characteristic of the IBD pathogen: the form of the virion is icosahedral, the size of the virion is 58-60 nm. Capsid T13, no shell.

Antigenic properties

According to its antigenic properties, the strain "K-58 No. 122 - DEPT" refers to serovariant I. The virus is stably neutralized by a homologous antiserum. The virus does not exhibit hemagglutinating activity. During vaccination, the virus induces the formation of specific antibodies detected by the RDP at a dilution of 1: 4-1: 64, in ELISA-1: 400-1: 10000 and more, with hyperimmunization in the RDP to 1: 4096 and higher, in ELISA> 1: 10000 . When determining the antigenic correspondence of the vaccine strain "K-58 No. 122 - DEPT" with field isolates of the IBD virus, a comparison was made of the variable region VP2 of 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 Taq DNA polymerase, the variable region of the VP2 gene encoding the main conformational epitope. 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 isolates of the IBD virus isolated at Russian poultry farms are antigenically different 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 "K-58 No. 122 - DEPT".

Biotechnological characteristics

The strain "K-58 No. 122 - DEPT" is intended for the manufacture of live and inactivated vaccines and diagnostic biologics. The strain "K-58 No. 122-DEPT" is reproduced in 9-11-day-old embryos of SPF-hens at a temperature of 37.7 ° C and a humidity of 55-64%. During 72-96 hours of incubation, the virus accumulates up to 5.5-7.0 lg EID _{50 / 0.2 cm} ³ .

Chemo- and genotaxonomic characterization

The strain "K-58 No. 122 - DEPT" is an RNA-containing virus with a molar mass of RNA of 2.16 × 10 ⁶ Da. Nucleic acid is 2-strand, 2-segment (A and B). The virus has a protein coat formed by 5 types of proteins (VP1, VP2, VP3, VP4 and VP5):

VP1 - RNA-dependent RNA polymerase;

VP2, VP3 - structural proteins;

VP4 - protease;

VP5 - not identified.

Lipids are absent.

The strain "K-58 No. 122 - DEPT" shows genetic stability. Most mutations are concentrated in the RNA region encoding VP2. Mutations leading to amino acid substitution determine the antigenic variability of VP2.

The strain "K-58 No. 122 - DEPT" shows 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.

Physical properties

The mass of the virion is - 55.0 × 10 ⁶ Da. Sedimentation coefficient 460S in sucrose gradient. The floating density of 1.33-1.34 g / cm ³ in the gradient of CsCl.

Resistance to external factors

The strain "K-58 No. 122-DEPT" is resistant to solvents (ether, chloroform) and detergents, sensitive to formaldehyde, UV radiation, γ-radiation and drying.

Additional features and properties

Immunogenic activity - 100%;

Reactogenicity - absent;

Pathogenicity is absent;

Virulence is absent;

Oncogenicity is absent;

Contagiousness is absent.

The strain "K-58 No. 122 - DEPT" is stable and harmless.

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 essence of the invention is illustrated by examples of its use.

Example 1

The viral replicate of the strain K-58 No. 122-DEPT on SPF-chicken embryos is obtained as follows. At the border of the membrana shell and chorioallantoic membrane (CAO), a notch is made without damaging the CAO, and then using a syringe, virus-containing material is introduced in an amount of 0.2 cm ³ per CAO. The hole in the shell is sealed with adhesive tape. Infected embryos are incubated at a temperature of 37.7 ° C for 48-120 hours. Fallen embryos are cut, frozen at a temperature of minus 40 ° C. The virus-containing material is thawed, crushed, extracted at 4 ° C for 45 minutes, frozen twice - thawed, phosphate buffer pH 7.5 is added, centrifuged for 20 minutes at 3000 rpm. The resulting virus-containing liquid is used as a matroe seed.

Example 2

Preparation of diagnostic antigen

SPF chickens 14-21 days old were inoculated with the strain K-58 No. 122-DEPT (intraocular, intranasal or oral). After 72 hours, chickens are killed, bursa are taken, frozen. 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 in the RDP activity of antigen prepared from strain "K-58 No. 122 - DEPT" are presented in table 1.

Example 3

Preparation of diagnostic serum

SPF - chickens 14-21 days of age are inoculated with an orally live vaccine from the strain K-58 No. 122-DEPT of the IBD virus. After 14 days, an inactivated antigen with an oil adjuvant is administered intramuscularly. After two weeks, the chickens are bled and receive hyperimmune serum, determining its activity in the RDP and ELISA. The results of serum studies are presented in tables 2 and 3.

Example 4

Preparation of dry diagnostic serum

SPF - chickens 14-21 days of age are injected with an orally live vaccine from the strain "K-58 No. 122 - DEPT" of the IBD virus. After 14 days, the chickens are inoculated with an intramuscularly inactivated antigen with an oil adjuvant, after 10 days the antigenic drug is administered again, and after 7 days the chickens are bled, receive serum and determine its activity.

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

Example 5

Inactivated vaccine preparation and testing

Inactivated vaccine against IBD birds from strain "K-58 No. 122 - DEPT" is prepared from a virus grown in developing embryos of SPF-chickens as described in example 1.

The virus is inactivated by aminoethylethyleneimine, adsorbed onto aluminum hydroxide with the addition of saponin.

The results of studies to determine the immunogenic activity of inactivated vaccines from strain "K-58 No. 122 - DEPT" are presented in table 5.

Example 6

Preparation of a live vaccine

At the first stage, matte material is prepared from the strain K-58 No. 122-DEPT of the IBD virus of birds as described in Example 1. The resulting virus-containing liquid is used for mass infection of 9-11-day-old SPF chicken embryos. Viral material in an amount of 0.2 cm ³ is introduced on HAO and incubated at a temperature of 37.7 ° C for 48-120 hours. Fallen embryos are cut, frozen at a temperature of minus 40 ° C and the obtained raw materials are used in the manufacture of the vaccine. The virus-containing material is thawed, crushed in a colloid mill, extracted for 45 minutes at 4 ° C, frozen twice - thawed, phosphate buffer pH 7.5 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 a temperature of minus 60-80 ° С. and lyophilized. The lyophilized material is corked under vacuum and tested for immunogenicity (in 14-day-old SPF chickens) and for safety (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 pink-brown suspension.

The vaccine is used in farms where clinical and pathological signs of the manifestation of IBD are recorded. Chicks are vaccinated from 10-14 days of age with an interval of 10-14 days at a dose of 100-1000 EID _{50 / cm} ³ method of feeding.

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

The resulting vaccine when titrated on 9-11-day old SPF chickens has a titer of 5.5-7.0 lg EID _{50 / 0.2 cm} ³ .

Example 7

Dry Live Vaccine Test

Determination of the immunogenic activity of the vaccine made as described in example 6 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 shown in table 6.

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

Example 8

Comparative evaluation of virus vaccines against IBD birds

In laboratory experiments and in the conditions of poultry farms of the Russian Federation, a comparative assessment of the immunogenic activity of domestic and imported live vaccines against IBD was carried out and it was shown that the dry vaccine against IBD from strain "BG" No. 102-DEPT is a more effective drug compared to other domestic and imported Therefore, the immunogenic activity of the strain "K-58 No. 122 - DEPT" was evaluated in comparison with the strain "BG" No. 102 - DEPT.

The results of a comparative assessment of the immunogenic activity of the strain "BG" No. 102 - DEPT "and strain" K-58 No. 122 - DEPT "are presented in table 7.

The data in table 7 indicate that the strain "K58 No. 122 - DEPT" is more reliable and effective, since it induces an antibody titer 1.5 times higher compared to the strain "BG" No. 102 - DEPT.

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

- strain "K-58 No. 122 - DEPT" of the IBD virus of 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;

- the strain "K-58 No. 122-DEPT" of the IBD virus obtained in accordance with the invention has, in comparison with the prototype strain, higher biological, antigenic and immunogenic activity in its native form and after inactivation and expands the arsenal of bird IBD virus strains, suitable for the manufacture of highly specific and sensitive diagnostics and highly immunogenic and harmless vaccine preparations.

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

Information sources

1. Syurin V.N. and coauthors. Viral diseases of animals. - M.: VNITIBP, 1998, 65-84.

2. Autosvid. USSR No. 1761802, C 12 N 7/00, 11/22/90

3. Winterfild R.W. Immunity response to the infectious bursal agent // Avian Dis., 1969, 13, 548-557.

4. Winterfild R.W. et al. Immune response and pathogenecity of different strains of infectious bursal disease virus applied as vaccines // Avian Dis., 1978, 22, 4, 721-731.

5. Winterfild R.W. et al. Characteristics of apparent derivates of the 2512 strain of infectious Bursal Disease virus when used as vaccines // Avian Dis., 1981, 25, 4, 900-910.

6. Wyeth P.F. The veterinary Record, 1979, 104, 188-193.

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Table 1 Determination of the activity of the IBB antigen from strain "K-58 No. 122-DEPT" in the RDP Part No. Activity Determination Method Antigen activity one RDP 1: 4 2 RDP 1: 4 3 RDP 1: 8 four RDP 1: 8 5 RDP 1: 8 6 RDP 1:16 7 RDP 1:16 8 RDP 1:16 9 RDP 1:16 10 RDP 1:16 table 2 Determination of serum activity No. of parties The name of the drug Adjuvant Antibody titer RDP IFA one live antigen inact. antigen oil 1: 1024 1: 7500 2 - - - 1: 1024 1: 9100 3 - - - 1: 1024 1: 10200 four - - - 1: 2048 1: 11500 5 - - - 1: 2048 1: 12300 6 - - - 1: 2048 1: 12500 7 - - - 1: 2048 1: 12850

Table 3 Determination of serum activity No. of parties The name of the drug Adjuvant Antibody titer RDP IFA one live antigen inact. antigen oil 1: 512 1: 9500 2 - - - 1: 1024 1: 9750 3 - - - 1: 1024 1: 10200 four - - - 1: 1024 1: 11500 5 - - - 1: 1024 1: 12080 6 - - - 1: 2048 1: 12200 7 - - - 1: 2048 1: 12800 Table 4 Determination of dry whey activity Part No. The name of the drug Adjuvant Antibody titer live antigen inactive. antigen inactive. antigen RDP IFA one - - - oil 1: 2048 1: 12500 2 - - - - 1: 2048 1: 13100 3 - - - - 1: 2048 1: 14000 four - - - - 1: 4096 1: 14500 5 - - - - 1: 4096 1: 15000 6 - - - - 1: 4096 1: 15300 7 - - - - 1: 4096 1: 16100

Table 5 Determination of the immunogenic activity of an inactivated vaccine against infectious bursal disease of birds from strain "K-58 No. 122-DEPT" No. of parties Name of material Antibody titer RDP IFA Vaccine Serum 1:16 1: 3500 one - 1:16 1: 3850 2 - 1:32 1: 4200 3 - 1:32 1: 4500 four - 1:32 1: 4850 5 - 1:64 1: 5200 6 - 1:64 1: 5370 7 - 1:64 1: 5600 8 - 1:64 1: 6100 9 - 1:64 1: 6500 10 - 1:64 1: 7220 Table 6 Immunogenic activity of dry vaccine against IBD birds from strain "K-58 No. 122-DEPT" №№ Vaccine dilution Vaccinated Control infection results infected got sick % save First experience one 10 ^-1 10 10 0 one hundred 2 10 ^-2 10 10 0 one hundred 3 10 ^-3 10 10 0 one hundred four 10 ^-4 10 10 one 90 5 The control 10 10 10 0 Second experience one 10 ^-1 10 10 0 one hundred 2 10 ^-2 10 10 0 one hundred 3 10 ^-3 10 10 0 one hundred four 10 ^-4 10 10 one 90 5 The control 10 10 10 0 Third experience one 10 ^-1 10 10 0 one hundred 2 10 ^-2 10 10 0 one hundred 3 10 ^-3 10 10 0 one hundred four 10 ^-4 10 10 one 90 5 The control 10 10 10 0

Table 7 Comparative evaluation of the immunogenicity of the strain "BG" No. 102-DEPT "and" K-58 No. 122-DEPT "virus IBD birds №№ The name of the strains "BG" No. 102-DEP "K-58№122-DEP" Antibody titer Avg titer Antibody titer Avg titer one 1: 7177 1: 8872 2 1: 2641 1: 7928 3 1: 5448 1: 5528 four 1: 3444 1: 5779 5 1: 3819 1: 4067 1: 8289 1: 6529 6 1: 2188 1: 8133 7 1: 2218 1: 2847 8 1: 4396 1: 7641 9 1: 1538 1: 3338 10 1: 7808 1: 6941

Claims (1)

The strain "K-58" of the virus of infectious bursal disease of birds (Bursitis infectiosa gallinae) of the family Birnaviridae, genus Avibirnavirus, serotype 1, a collection of FSI VGNKI "K-58 No. 122-DEPT", for the manufacture of diagnostic and vaccine preparations.