The strain of virus infectious laryngotracheitis birds for the manufacture of diagnostic and vaccine preparations

 

The invention relates to veterinary Virology and biotechnology. The original virus to obtain strain "On" selected from the larynx and trachea chicken with signs of a latent form of the disease in Kazakhstan in 1992, the Production strain "On" is received by multiple serial passages in 9-11-day-old embryos SPF chickens. The strain is deposited in the Collection of microorganisms VGNKI 05.06.2000, under the registration name ON 112-DEPT". The strain shows high biological, immunogenic activity as in the native form and after inactivation. The strain is cultivated in 9-11-day-old embryos SPF chickens, and 3-6 days of cultivation he reaches the titer of infectivity at least 6.2 Ig EID50/cm3. The strain has a high biological, antigenic and immunogenic activity in native and inaktivirovannoj and suitable for the manufacture of highly specific and sensitive diagnostic and highly immunogenic, safe and reactogenic vaccine drugs for ocular and oral methods of immunization. 6 table.

The invention relates to the field of veterinary Virology and biotechnology and can be used in the development and production of religious disease of birds is the reason for the high percentage of mortality (normal mortality is 10-20% of the known cases of high mortality, which accounts for 50-70%), we recover individuals reduced daily gain and egg production. TLI is called the herpes virus. The complex responses to TLI one of the main tools is vaccination with live and inactivated vaccines.

The advantages of virus-vaccines are that they are suitable for mass application by watering, aerosol spray, etc. for immunization of birds requires a relatively small amount of immunizing drug, as a live antigen continues to multiply in the body immunized birds, they are cheaper to manufacture and to store them requires relatively little space in refrigeration.

In world practice there are more than 20 strains of the virus TLI birds used for the manufacture of vaccines (1-6). Known strains of the virus TLI differ in virulence for birds and chick embryos (CE), tropism, the rate of release from cells in tissue culture, stability during storage, the avidity, molecular structural features, deviations strains of different virulence in the pic is the manufacture of vaccines (10).

A known strain "SNIIP virus TLI birds used for the manufacture of vaccines (3).

Known clone "NT" strain "SNIIP virus TLI birds used for the manufacture of vaccines (11).

The disadvantages of the known strains are in their low immunogenic activity. Vaccines of these strains form the immune system, which is not able to protect 100% ptitsepogolovya from infection with epizootic virus TLI.

The closest to the proposed invention, the essential features is the strain VNIIMP virus TLI birds used for the manufacture of vaccines (12).

Disadvantages strain of the prototype are its low immunogenic activity and increased reactogenicity.

Given the high information capacity of the viral genome TLI and diversity of its phenotypic properties, relevant problem is the search for new vaccine strains of this pathogen with high biological, immunogenic and antigenic activity, both in native and in inaktivirovannoj, and low reactogenicity.

In the task of creating the present invention was to enlarge the Arsenal of virus strains TLI with high biological immune is isotopentechnik and sensitive diagnostics and highly immunogenic, harmless and reactogenic vaccine drugs for ocular and oral methods of immunization.

The technical result from use of the present invention is to expand the Arsenal of virus strains TLI birds with high biological, immunogenic and antigenic activity both in native and in inaktivirovannoj and suitable for the manufacture of highly specific and sensitive diagnostic and highly immunogenic, safe and erectogenic vaccine drugs for ocular and oral methods of immunization.

This technical result is achieved by receiving the attenuated strain Of virus TLI birds. The strain is new, previously unknown. The original virus to obtain strain "On" selected from the larynx and trachea chicken with signs of a latent form of the disease in Kazakhstan in 1992. Production strain "On" is received by multiple serial passages in 9-11-day-old embryos SPF chickens.

Strain "On" deposited in the all-Russian state collection of strains of microorganisms used in veterinary medicine and animal husbandry, all-Russian state research Institute for control, standardisation and certificaat high biological, antigenic and immunogenic activity in the native form and after inactivation. Experimentally confirmed the possibility of its use for the manufacture of diagnostic and vaccine preparations.

The strain Of virus TLI birds characterized by the following features and properties.

Morphological properties of the Strain Of virus TLI belongs to the family Herpesviridae. When electron microscopy (instrumental increased 4.5 x 105and 5 x 105in the field of view of the microscope see distinct viral particles TLI characterized by the morphology. Virions are spherical in shape, and their sizes vary from 200 to 250 nm (some up to 330 nm). Virions TLI consist of spherical nucleocapsid surrounded supercasino membrane complex shape. The virion has an icosahedral symmetry. The icosahedral capsid is covered lipoproteidna shell having on its surface morphological units-Palomera. The diameter of the nucleocapsid 1132 nm, the average diameter of nucleocapside shell about 350 nm.

Nucleocapside size 100-110 nm are arranged in a viral particle, usually eccentric, rarely in the center of the particles. Virus genome TLI has a length 155000 polynucleotides s (17000 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 virus TLI.

Antigenic properties of the Strain Of virus TLI consistently neutralized homologous serum, is identified in the blood serum of chickens, containing antibodies against this virus, RDP, PH and ELISA. In RDP identified homologous hyperimmune sera in titer of 1:8. In the PH index neutralize homologous hyperimmune serum in dilutions 1: 10 and 1:20 average of 3.8 Ig EID50/cm3respectively. In ELISA identified field and laboratory hyperimmune sera in titer to 1:24579. Antigenic differences between strains of "O" relative to other strains TLI is not installed. Hemagglutination properties strain does not possess.

Biotechnological characteristics of Strain "On" shows high biological, immunogenic and antigenic activity as in the native form and after inactivation. Strain "On" is used for the manufacture of live and inactivated vaccine against ILT birds and diagnostic products. The virus has a pronounced tropism for epithelial cells of the mucous membrane of the larynx, trachea and cloaca of birds. The strain is cultivated in 9-11-day-EMBs 10-20% suspension HAO on the 5th day of cultivation, provided a generalized distribution across the membrane is at least 6.2 Ig EID50/cm3.

Chemo - and geotectonically characteristic Strain "On" is a DNA-containing virus.

Resistance to external factors the Virus is sensitive to lipolytic agents, heat and various disinfectants. Temperature 55oWith destroys the virus for 10-15 minutes and 38oWith over 48 hours In the frozen carcasses of sick and forced killed chickens, kept at a temperature of minus 10, 18 and 28oWith, the virus remained active for more than 19 months, on the surface of the shell in terms of thermostat is inactivated after 12 h, and infected poultry is kept not more than 9 days. Fully inactivated in 1% solution of Creole within 30 seconds long He remains active in liofilizirovannom state or minus 20-60oS. liofilizirovannom homogenate HAO virus retains infectivity at least 1.5 years. In frozen tissue HAO virus infectivity is retained at least 4 years.

The immunogenicity of the strain When the vaccination of chickens older than 15 days old ocular method at a dose of 1000 EID50and oral way (feeding) at a dose of 4000 EID50induces the production of specific antibodies and creates immunity against TLI at 100% of the vaccinated chickens. In liofilizirovannom homogenate HAO virus bahraintane bird transmission of vaccine virus strain "On" contact is not installed.

Conditions for maintaining the strain Liofilizovannye material from strain "On" stored at a temperature of 4oWith, vaccinated HAO - at a temperature not higher than minus 40oC. For lyophilization using stabilizer "ARRIAH". For resuspendable of liofilizirovannogo virus using isotonic phosphate buffer solution pH 7.4 and 7.6.

Genetic purity After 20 passages on HAO 9-11-day-old embryos SPF-chickens any changes immunobiological properties of strain "On" is not set.

Additional characteristics and properties
Reactogenicity is missing.

Pathogenicity is missing.

Virulence is missing.

Carcinogenesis is missing.

Contagiousness is missing.

The stability of attenuation noted when conducting 4 passages in susceptible chickens.

On the basis of the obtained data it can be argued that the strain Of virus TLI on antigenic and immunological spectra is original, in taxonomic relation to new, previously unknown variant of the virus TLI.

According to the applicant, the proposed strain corresponds to the conditions paternopoli "novelty" and "inventive step".

The essence of the invention is illustrated by examples of its ispor> In shell chicken embryos from the air chamber to form a hole with a diameter of 8-10 mm In Podkolodny shell and HAO injection needle produce four puncture in the corners of the imaginary square with a diagonal of about 5 mm, then put vaccinated material from strain "On" in the amount of 0.1 cm3. The hole in the shell stick with adhesive tape. Infected embryos incubated for 5 nights, 6 days determine the characteristic of the virus TLI defeat HAO. From the affected HAO cook 15-30% suspension, which proparaguay and free from coarse particles by low-speed centrifugation. Received vaccinated fluid with biological activity not less than 6,0 Ig EID50/cm3used as motroway seed for mass infection 9-11-day-old embryos SPF chickens.

EXAMPLE 2.

Virus-vaccine against ILT birds from the attenuated strain of "About" is prepared as follows.

At the first stage of preparing matrawy material from the production strain Of virus TLI birds as described in example 1. Vaccinated suspension obtained from the affected HAO, used for mass infection 9-11-day-old embryos SPF chickens. Infection of embryos will produce precolombino for 3-6 days at 37,7oC and humidity 60%. According to the results of ovoscope and subsequent autopsy to determine the presence of typical virus TLI defeat HAO. Affected HAO used as vaccinated material. From the affected HAO cook 15-30% suspension, which proparaguay and free from coarse particles by low-speed centrifugation. Vaccinated supernatant liquid is carefully poured into sterile conditions, and then it added the stabilizer, which can be used (separately or in combination) skim milk, lactose, gelatos, sucrose and other Vaccinated liquid and a stabilizer are mixed in a ratio of at least 17:3-19:1. The resulting mixture was filled into vials proparaguay at a temperature of minus 60-80oWith and subjected to lyophilization. Liofilizovannye material sealed under vacuum and subjected to examination for immunogenic activity of ocular and oral methods of vaccination 1000 and 4000 EID50respectively, and emissions ocular and oral methods of inoculation on 10000 and 40000 EID50respectively.

Received the vaccine during the titration of 10-day-old embryos SPF chickens has a titer of from 6.2 to 6.6 Ig EID50/cm3.

the first has the form of a homogeneous porous structure beige color in the form of cylindrical pellets, soluble in water or a special solvent.

Before applying the virus the vaccine is diluted with a diluent, which represents a 5% solution of glycerol in phosphate-buffered solution, marked neutral food coloring, type, Indigo Carmine, and is intended to prepare the working dilution of the vaccine in ocular method of immunizing poultry and bringing the pH of the water is close to neutral or slightly alkaline, with oral method of immunization.

Vaccination is subject to clinically healthy bird older than 15 days of age. Bird up to 60 days age Vaccinium twice with an interval of 14 days. Immunity is formed through 7 days after revaccination. Re-vaccination is carried out every 6 months.

EXAMPLE 3.

Tested immunogenic virus-vaccine against ILT birds, made as described in example 2. The definition of immunogenic vaccine was conducted in chickens. For this pre-prepared dilution of the vaccine in the diluent: for ocular method of immunization so that the 0.05 cm3(the volume of one drop) contained 1000 EID50for oral - to 1 cm3contained 4000 EID50. In the experience took 50 chickens and role. After 18 days after vaccination in experimental and control chickens were infected by the virulent strain of "Bogatischevskaya virus TLI birds. The virus was injected intratrahealno at a dose of 500 EID50in the amount of 0.5 cm3. For chickens were observed for 10 days. The vaccine is immunogenic, if it protects from disease TLI 16 chickens of 20 vaccinated. In the control group should be ill with signs of ILT at least 8 chickens from 10 infected. The results are given in table 1, which indicate that all three prepared a series of virus-vaccine against ILT birds from strain "On" for all tested parameters correspond to the requirements laid down in the technical specifications for the preparation and almost equally effective for ocular and oral application on the bird. In the control group of chickens, the incidence of TLI was 100%, and the mortality at 3 experiments amounted to 48.6%.

EXAMPLE 4.

Inactivated vaccine against ILT is prepared from a strain Of virus grown in the developing embryos SPF chickens as described in example 2. The resulting virus inactivating aminoethylethanolamine, adsorb on Aerosil A-300 with the addition of saponin. The results of studies to determine the immunogenic inwat as follows. SPF-chickens 25-30 days of age inoculant oral or ocular live vaccine of the strain Of virus TLI. After 14 days all chickens injected intramuscularly inactivated antigen with an oil adjuvant, and two weeks later the chickens bleed and get Hyper active serum and determine its activity in the RDP and ELISA. The results are shown in table. 3.

EXAMPLE 6.

Diagnostic serum prepared as follows. SPF-chickens 25-30 days of age administered orally or ocular live vaccine of the strain Of virus TLI. After 14 days all chickens inoculant intramuscular inactivated antigen, using as adjuvant hydrophobic Aerosil or hydrate of aluminum oxide. After two weeks the chickens bleed, get the serum and determine its activity in the RDP and ELISA. The results are shown in table. 4.

EXAMPLE 7.

Dry diagnostic serum prepared as follows. SPF-chickens 25-30 days of age, type of oral live vaccine of the strain Of virus TLI. After 21 days, all Chicks inoculant intramuscular inactivated antigen with an oil adjuvant. After 7 days an inactivated antigen is administered repeatedly and after 7 days the Chicks on the substance additives and subjected to lyophilization. Liofilizovannye serum used for ELISA test. Research results serum are presented in table. 5.

EXAMPLE 8.

Comparative evaluation of immunogenic virus-vaccine against ILT birds, prepared from strains of domestic and foreign origin: virus vaccines "Laryngo-vac" (Holland), virus-vaccines clone "NT" strain "SNIIP" virus-vaccine from strain "VNIIBT and virus-vaccine from strain "On". To study the efficacy of these vaccines was conducted in SPF chickens 25 days age. Chickens were injected with one dose of the tested vaccines oral and ocular. Assessment of vaccine effectiveness was carried out according to the control of infection with the virulent strain "Bogatischevskaya virus TLI. The results of these studies are presented in table. 6.

The data table. 6 indicate that when controlling infection in vaccinated chickens with virulent strain of "Bogatischevskaya virus TLI only virus vaccine from strain "On" induced the formation of immunity to TLI in 100% of vaccinated chickens.

Thus, the above information shows the implementation of the use of the present invention, the following cumulative conditions:
the strain Of virus Ieronymos of Virology and biotechnology;
the strain Of virus TLI received in accordance with the invention, it has high biological, antigenic and immunogenic activity in the native form and after inactivation and suitable for the manufacture of diagnostics and vaccines;
for the present invention in the form as it is described in the independent claim, confirmed the possibility of its implementation using the steps described in the application or known before the priority date tools and methods.

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

Sources of information taken into account when preparing the description of the invention the application for the grant of a patent of the Russian Federation for the invention of "Strain "On" virus TLI birds for the manufacture of diagnostic and vaccine preparations.

Sources
1. U.S. patent 3331736; 167-78, 18.07.67,

2. U.S. patent 3444293; 167-78, 13.05.69,

3. Auth. mon. The USSR 129791; 30h, 6; 1960

4. Auth. mon. The USSR 1750683 And 61 To 39/245, 30.07.92,

5. RF patent 2138290 And 61 To 39/245, 39/265, 35/78; 27.09.99,

6. Syurin C. N., Samuyilenko A. Ya. and other Viral diseases of animals. M, UNITEMP, 1998, 672-683.

7. Glissen J. R. Proceedings, 1988, 137.

8. Goodwin, M. A. et al. Avian Diseases, 1995, 39, 2,444.

9. Peixuan Guo et al. Viro Y. S., Shevchenko, A. A. and other Immunological efficacy virus-vaccine against infectious laryngotracheitis chickens. Veterinary medicine, 1991, 3, 32-34.

12. Malosco centuries, Sokolov, C. D. and others experience in the fight against infectious laryngotracheitis. Poultry, 1983, 12, 21-23 (prototype).


Claims

The strain of the virus of infectious laryngotracheitis birds, SEM. Herpesviridae, genus Herpesvirus, collection VGNKI "ABOUT 112-DEPT" for the manufacture of diagnostic and vaccine preparations.

 

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3 dwg, 1 ex

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