RU2378014C2 - Emulsion inactivated associated vaccine against cattle paragrippe-3, rednose and coronoviral infection - Google Patents

Abstract

FIELD: veterinary science.

SUBSTANCE: invention relates to veterinary virology and biotechnology. The vaccine contains an active substance and a target additive. The vaccine active substance is represented by a mixture of avirulent refined antigen material from the VGNKI-4 strain of cattle paragrippe-3 virus, Paramyxoviridae family, Parainfluenza genus, Parainfluenza virus 3 subtype, collection of Federal State Institution "Russian National Research Institute for Control, Standardisation and Certification of Veterinary Preparations", "VGNI-4 DEP", avirulent refined antigen material from the VNIIZZh strain of cattle rednose virus, Herpesviridae family, Varicellavirus genus, collection of Federal State Institution "Russian National Research Institute for Control, Standardisation and Certification of Veterinary Preparations", "VNIIZZh-DEP", and of avirulent refined antigen material from the VNIIZZh strain of cattle coronavirus, Coronaviridae family, Coronavirus genus, collection of Federal State Institution "Russian National Research Institute for Control, Standardisation and Certification of Veterinary Preparations", "VNIIZZh-DEP". The strains are taken at a ratio of 1: 1: 1 and in quantities ensuring protective immune activity of each antigen in the animal organism after receipt of the target preparation injection.

EFFECT: with the animals having been immunised the vaccine induces a high level of antigens; immunity with them is generated within 10-15 days after the vaccine re-injection and sustains for at least 6 months.

15 cl, 7 tbl, 4 ex

Description

The invention relates to the field of veterinary virology and biotechnology and relates to an associated vaccine for the specific prophylaxis of respiratory and gastrointestinal diseases of cattle (cattle) caused by pathogens of parainfluenza-3 (PG-3), infectious rhinotracheitis (IRT) and coronavirus infection of cattle.

These viruses play a leading role in the etiology of respiratory and gastrointestinal diseases of cattle, especially in large livestock farms.

PG-3 (cattle transport fever, parainfluenza-3) is an acute contagious viral disease, mainly calves, characterized by respiratory damage, but the latent form of parainfluenza infection is most characteristic. PG-3 occurs in cattle, both in the form of mono-infection and in the form of mixed viral and viral-bacterial infections, which cause significant economic damage due to the death and culling of animals, reduced productivity and impaired reproduction. PG-3 is ubiquitous, in almost all countries of the world.

IRT is an acute viral contagious disease of cattle, causing significant economic damage to livestock.

IRT virus causes cattle disease of various age groups, manifested in five forms: damage to the upper respiratory tract, vaginitis, encephalitis, conjunctivitis and arthritis. In addition, pneumonia is possible in calves.

The economic damage caused by ИРТ consists of a decrease in milk yield during the period of illness (up to 50-60%), a significant increase in the service period and the barrenness of cows with a vaginal form, poor development of sick young animals and a significant percentage of death and culling of calves. In chronic serous-purulent pneumonia, up to 20% of young animals die. The disease is widespread throughout the world.

Coronavirus infection in cattle is manifested in the form of coronavirus diarrhea in newborn calves, coronavirus winter dysentery of adult cattle, and coronavirus respiratory infection.

Extremely widespread, the tendency to cause long-term persistence, and relative resistance to various factors determine the enzootic course of the disease in farms with poor living and feeding conditions. The stationarity of the disease is maintained due to the prolonged persistence of the pathogen in the body of animals-convalescents.

These infections occur in cattle, both in the form of mono-infections and in mixed viral and viral-bacterial infections.

Currently, the main means of combating respiratory and gastrointestinal diseases of cattle is specific prophylaxis, for which live and inactivated vaccines are used in the form of mono- and associated drugs (1, 2, 3).

The advantage of live vaccines due to manufacturability and ease of use is beyond doubt. However, the immune response in animals when they are introduced is not always stable and in some cases causes complications after vaccination: the vaccine can cause increased reactogenicity, immunosuppression, and sometimes death of up to 2% of animals during vaccination, depending on the conditions of detention and the presence of co-infections, reproduction problems, prolonged persistence and isolation of the vaccine virus into the environment. For this reason, they are not widely used.

For specific prophylaxis of these diseases, inactivated vaccines, both sorbed and emulsion, are widely used.

A known vaccine against cattle PG-3 adsorbed inactivated containing avirulent purified antigenic material from the strain VGNKI-4 of the PG-3 virus obtained in the culture of BHK-21 cells with an infectious activity of at least 6.5 Ig TCD ₅₀ / cm ³ , and as the target additive adjuvant-sorbent aluminum hydroxide (GOA) in an effective ratio (4).

A known vaccine against cattle ИРТ is inactivated emulsion containing avirulent purified antigenic material from the ARRIAH strain of cattle ИРТ virus obtained in an animal cell culture taken from the group consisting of BHK-21 cell culture, MDBK cell culture and from a culture of ZKG cells, with an infectious activity of at least 6.0 Ig TCD ₅₀ / cm ³ and activity in the RDP of at least 4.0 log, and as a target additive, an oil adjuvant in the ratio, wt.%:

Antigenic material 30,0 Oil adjuvant up to 100.0 (5)

A known vaccine against coronavirus infection of cattle is adsorbed inactivated, containing as an active substance an avirulent purified antigenic material from the ARRIAH strain of cattle coronavirus obtained in animal cell culture, taken from the group consisting of MDBK cell culture, Taurus-1 cell culture, from KST cell culture and from Vero cell culture, in the concentration of viral particles, which provides protective immune activity of the antigen in the animal’s body after administration of the target drug as a target additive adjuvant GOA-sorbent in an effective ratio (6).

The disadvantages of monovalent inactivated vaccines are that they stimulate the formation of immunity in animals to only one infection, require prophylactic treatments against each infection separately, which affects labor costs, stretches the formation of immunity in animals, and contributes to the occurrence of stressful phenomena and complications.

These disadvantages are partially eliminated by the creation of associated vaccines containing separately or in combination antigens of pathogens PG-3, IRT and coronavirus infection of cattle of various strains.

In some cases, the composition of known vaccines, especially designed to protect calves, include antigens of pathogens that cause secondary infections.

An associated vaccine is known for viral diarrhea — mucosal disease (VD-BS), rota- and coronavirus infections, and cattle escherichiosis — inactivated emulsion containing a mixture of avirulent purified antigenic materials from strain VDV-Oregon-C24 V (CAPM V-293) as an active substance. VD-BS virus of cattle, from strain TM-91 (CAPM V-279) of rotavirus cattle, from strain BCV-9 (CAPM V-280) of coronavirus cattle and from strains 3935/1 (CAPM 6233), 4804 (CAPM 6234) and 6304 (CAPM 6235) E. coli bacteria obtained in sensitive biological systems, and oil ad Juvent in effective ratios (7).

Known associated vaccine against IRT, rota- and coronavirus infections and Escherichia cattle inactivated emulsion, containing as an active substance a mixture of avirulent purified antigenic materials from strain IBR-BK-5-PTOB-8 INRV No. 3760 of cattle IRT virus, from strain TM-91 (CAPM V-279) cattle rotavirus, from strain BCV-9 (CAPM V-280) of coronavirus cattle and from strains 3935/1 (CAPM 6234) and 6304 (CAPM 6235) of E. coli bacteria obtained in sensitive biological systems, and as the target additive, an oil adjuvant in effective ratios (8).

Known vaccine associated PG-3 rotavirus and coronavirus infections and ehsherihioza RNC inactivated emulsion comprising as active ingredient the mixture of purified antigenic material avirulent strain of PI-3 / T _1/11 V-30, PG-3 virus RNC from strain TM-91 (CAPM V-279) cattle rotavirus, from strain BCV-9 (CAPM V-280) cattle coronavirus and from strains 3935/1 (CAPM 6233), 4808 (CAPM 6234) and 6304 (CAPM 6235) E. bacteria coli obtained in sensitive biological systems and, as a target additive, an oil adjuvant in effective ratios (9).

An associated vaccine against rota-, corona-, herpesvirus and Escherichia diarrhea of newborn calves is known, inactivated adsorbed, containing as an active substance a mixture of avirulent purified antigenic materials from Bovine rotavirus strain III-1, from Bovine coronavirus strain CM-1, from strain TKA-VIEV-V-2 "Bovine Herpesvirus and from strains of bacteria E. coli containing adhesins K ₉₉ and A ₂₀ , and as a target additive adjuvant-sorbent GOA in effective ratios (10).

An associated vaccine against rota- and coronavirus infections, Escherichiosis and clostridiosis of cattle is known, inactivated emulsion containing a mixture of avirulent purified antigenic materials of their strains Cody 81-4, 10B223 of type G and B 641 of cattle rotavirus, Mebus strain (ATCC No. VR- 874) cattle coronavirus, strains B41, B43, B44 and B141 of E. coli bacteria and strain GL 47 (ATCC PTA-3000) of bacteria Clostridium perfringens obtained in sensitive biological systems, and as the target additive, an oil adjuvant in effective ratios (11) .

Known associated vaccine against IRT, PG-3, viral diarrhea, respiratory syncytial infection, rota- and coronavirus diseases of calves "COMBOVAC" inactivated adsorbed containing the mixture of avirulent purified antigenic materials from 6 production virus strains as an active substance: IRT, PG- 3, VD-BS, respiratory syncytial infection, rota- and coronavirus diseases of cattle, obtained in sensitive biological systems, and as a target additive adjuvant-sorbent GOA in an effective ratio niyah (12).

Known associated vaccine against viral pneumogastroenteritis of cattle and calves is inactivated adsorbed, containing as an active substance a mixture of avirulent purified antigenic materials from strains B-25 and TK-A of the IRT virus of cattle, from strains VD-N and VD-R of the virus VD-BS cattle, from strains B-19 and PTK-45/86 of the PG-3 cattle virus, from strains K-88 and RP-82 of cattle rotavirus, from strains KV-90 and K-22 of cattle coronavirus, and from strains PC-89 and PC-71 causative agent of respiratory syncytial infection of cattle obtained in sensitive biological systems, and as targets minutes additive adjuvant sorbent GOA in effective ratios (13).

There is also a method for the quantitative assessment of virus-containing suspensions using electron microscopy, which arose as a result of many years of experiments on electron-microscopic control of viral preparations and comparison of its data with indicators of biological tests, for example, virus infectivity titer (14).

The authors of this method established the relationship between the infectivity titer of native FMD virus suspensions and the number of physical virus particles observed under an electron microscope, which served as the basis for determining the total concentration of viral particles per unit volume of the suspension (the degree of accumulation of viral particles in a certain volume of liquid).

To determine the total concentration (N) of viral particles per unit volume of the analyzed suspension on the screen of an electron microscope with an area of S ₂ count the number of simultaneously observed particles n. The well-known instrumental increase in the electron microscope (M) determines the size of the portion of the substrate film S ₁ corresponding to the useful screen area:

When applying viral preparations to the substrate films, the concentration of viral particles (n) in this area S ₁ corresponds to the total amount of adsorbed particles and particles deposited on the substrate during evaporation of the residual liquid. Therefore, the number of particles observed simultaneously on the screen is distributed in a microvolume equal to:

v = S ₁ · I,

Where

I is the thickness of the residual liquid layer on the substrate (mm).

In the determined volume of the virus-containing suspension, the number of particles is equal to:

,

,

and after replacing the values of v and S ₁ :

,

,

Where

V is the volume of the analyzed suspension (1 cm ³ );

N is the arithmetic average of a series of values of the number of particles calculated from the screen of an electron microscope or exposed photographic plates.

The thickness of the residual layer (I) on the substrate is measured by a pre-cooled micrometer after freezing the liquid on the substrate in liquid nitrogen vapor. It is 0.03-0.04 mm and it is taken as a constant value.

Electron-microscopic determination of the concentration of physical viral particles allows not only to determine the quantitative characteristic of the virus in the analyzed suspensions, but also to assess the morphological state of the studied viral particles.

The specified method turned out to be universal and suitable for determining the quantitative characteristics of various viruses in the studied liquids.

The closest to the proposed invention, the essential features is associated vaccine PG-3 and coronavirus RNC infection emulsion inactivated, comprising as active ingredient the mixture avirulent purified antigenic material from a strain of PI-3 / T _1/11 V-30 virus GHG-3 Cattle from the BCV-9 strain (CAPM V-280) of cattle coronavirus obtained in sensitive biological systems and, as a target additive, an oil adjuvant in effective ratios (9).

A common significant drawback of the known associated vaccines, including the prototype vaccine, intended for the prevention of massive infectious respiratory and gastrointestinal diseases of cattle of various age and gender groups, is their insufficiently high efficiency due to:

1) inferiority (incompleteness) of the antigenic composition of their constituent ingredients and complex interactions of the antigens from which they are made;

2) the narrowness of the antigenic spectrum of action of the strains used;

3) low immunogenic activity of drugs. The reason for this drawback may be the use of formalin for inactivation of viruses, which causes destructive changes in viral proteins.

Therefore, the problem of creating an associated vaccine against the main pathogens of infectious respiratory and gastrointestinal diseases of cattle and calves continues to be relevant and is the main direction of research to improve them.

The problem to which the present invention is directed, is to obtain an associated vaccine against PG-3, IRT and coronavirus infection of cattle emulsion inactivated, which creates effective protection of susceptible animals of various sex and age groups against pathogens PG-3, IRT and coronavirus infection of cattle circulating in the territory Russian Federation.

The technical result from the use of the present invention is to expand the arsenal of highly immunogenic, harmless and areactogenic associated vaccines against PG-3, IRT and coronavirus infection of cattle emulsion inactivated, capable of protecting livestock of various sex and age groups from pathogens PG-3, IRT and coronavirus infection of cattle on Russian territory.

The specified technical result was achieved by the creation of an associated vaccine against PG-3, IRT and coronavirus infection of cattle with inactivated emulsion, characterized by the following set of features. According to the invention, the vaccine according to the invention contains a mixture of avirulent purified antigenic materials from the strain “VGNKI-4” (copyright name) of the cattle PG-3 virus, from the strain “ARRIAH” (copyright name) of the IRT virus and from the strain “ARRIAH” (author's name) cattle coronavirus obtained in cell cultures of animal origin, in quantities that ensure the protective immune activity of each antigen in the animal’s body after administration of the target drug to it, and as a target additive m slyany adjuvant in an effective ratio.

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

1. Associated vaccine against PG-3, IRT and coronavirus infection of cattle emulsion inactivated.

2. The active substance.

3. As an active substance, a mixture of avirulent purified antigenic materials from strain VGNKI-4 of the PG-3 cattle virus, from the strain ARRIAH of the IRT virus of cattle and from the strain ARRIAH of cattle coronavirus obtained in animal cell cultures in amounts providing protective immune activity of each antigen in the animal’s body after administration of the target drug to it.

4. Targeted supplement.

5. As a target additive, an oil adjuvant.

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. Associated vaccine against PG-3, IRT and coronavirus infection of cattle emulsion inactivated.

2. The active substance.

3. Targeted supplement.

Compared with the prototype vaccine, the essential distinguishing features of the proposed vaccine are that it contains a mixture of avirulent purified antigenic materials from the strain VGNKI-4 of the PG-3 virus, from the strain ARRIAH of the IRT virus and from the strain " ARRIAH "of cattle coronavirus obtained in cell cultures of animal origin, in amounts that provide protective immune activity of each antigen in the animal’s body after administration of the target drug to it.

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

1. Avirulent purified antigenic material from VGNKI-4 strain of PG-3 cattle virus obtained in a cell culture of animal origin, taken from the group consisting of MDBK calf kidney cell culture and VNK-21 Syrian hamster kidney cell culture quantity.

2. Avirulent purified antigenic material from the VGNKI-4 strain of PG-3 cattle virus obtained in MDBK cell culture with an infectious activity of at least 6.5 Ig TCD ₅₀ / cm ³ , in an amount of at least 10.0 wt.%.

3. Avirulent purified antigenic material from the VGNKI-4 strain of PG-3 cattle virus obtained in the BHK-21 cell culture with an infectious activity of at least 6.5 Ig TCD ₅₀ / cm ³ , in an amount of at least 10.0 wt. %

4. Avirulent purified antigenic material from the ARRIAH strain of cattle IRT virus obtained in a cell culture of animal origin, taken from the group consisting of a cell culture of BHK-21, from a cell culture of MDBK and from a gonad cell culture of 3-day goat 3KG, in effective amount.

5. Avirulent purified antigenic material from the ARRIAH strain of cattle IRT virus obtained in VNK-21 cell culture with an infectious activity of at least 6.0 Ig TCD ₅₀ / cm ³ and activity in the diffusion precipitation reaction (RDP) of at least 4.0 log ₂ , in an amount of not less than 10.0 wt.%.

6. Avirulent purified antigenic material from the ARRIAH strain of cattle IRT virus obtained in MDBK cell culture with an infectious activity of at least 6.0 Ig TCD ₅₀ / cm ³ and activity in the RDP of at least 4.0 log ₂ , in an amount of at least 10.0 wt.%.

7. Avirulent purified antigenic material from the ARRIAH strain of cattle IRT virus obtained in the culture of cells of the GCC with an infectious activity of at least 6.0 Ig TCD ₅₀ / cm ³ and activity in the RDP of at least 4.0 log ₂ , in an amount of not less than 10.0 wt.%.

8. Avirulent purified antigenic material from the ARRIAH strain of cattle coronavirus obtained in a cell culture of animal origin, taken from the group consisting of MDBK cell culture, Taurus-1 calf kidney cell culture, KST calf coronary cell culture, and culture Vero monkey kidney cells in an effective amount.

9. Avirulent purified antigenic material from the ARRIAH strain of cattle coronavirus obtained in an MDBK cell culture with a degree of accumulation of at least 10 ⁸ virus particles / cm ³ and activity in the hemagglutination reaction (RGA) of at least 7.0 log ₂ , not less than 10.0 wt.%.

10. Avirulent purified antigenic material from the ARRIAH strain of cattle coronavirus obtained in Taurus-1 cell culture with a degree of accumulation of at least 10 ⁹ viral particles / cm ³ and activity in the RGA of at least 9.0 log ₂ , in an amount of at least 10 , 0 wt.%.

11. Avirulent purified antigenic material from the ARRIAH strain of cattle coronavirus obtained in a culture of KST cells with an accumulation degree of at least 10 ^7.5 virus particles / cm ³ and activity in the RGA of at least 6.0 log ₂ , in an amount of at least 10 , 0 wt.%.

12. Avirulent purified antigenic material from the ARRIAH strain of cattle coronavirus obtained in Vero cell culture with an accumulation degree of at least 10 ^7.5 virus particles / cm ³ and activity in the RGA of at least 6.0 log ₂ , in an amount of at least 10 , 0 wt.%.

13. Oil adjuvant in an amount of not less than 70.0 wt.%.

14. A mixture of avirulent purified antigenic materials from the strain VGNKI-4 of the PG-3 virus, from the strain ARRIAH of the IRT virus and from the strain ARRIAH of cattle coronavirus obtained in animal cell cultures, and an oil adjuvant in the ratio, wt. %:

Avirulent purified antigenic material from strain

"VGNKI-4" virus PG-3 cattle obtained in cell culture

animal origin taken from the group consisting of

cell cultures of MDBK and from the cell culture of BHK-21, with an infectious activity of at least 6.5 Iq TCD ₅₀ / cm ³ - 10.0

Avirulent purified antigenic material from strain

ARRIAH of cattle IRT virus obtained in cell culture

animal origin taken from the group consisting of

BHK-21 cell culture, from MDBK cell culture and from

ZKG cell culture, with infectious activity of at least 6.0 Ig TCD ₅₀ / cm ³ and activity in the RDP of at least 4.0 log ₂ - 10.0

Avirulent purified antigenic material from strain

ARRIAH of cattle coronavirus obtained in cell culture

animal origin taken from the group consisting of

cell culture MDBK, from cell culture Taurus-1,

from a culture of CTC cells and from a culture of Vero cells,

in the concentration of viral particles, providing

protective immune activity of the antigen in the animal’s body after administration of the target drug to it - 10.0 Oil adjuvant - up to 100.0,

The virus strains included in the proposed vaccine are known and are industrial in the manufacture of monovaccines (15, 16, 17).

As a result of studies, the authors found that between the antigens that make up the proposed vaccine, there is no competition. The authors also discovered the phenomenon of synergism when combining these antigens in the proposed vaccine, which allowed to reduce their quantitative content in the drug while maintaining the same tension and duration of immunity in vaccinated animals, as when using the corresponding monopreparations for immunization.

An additional technical result from the use of the present invention is achieved due to the fact that for the inactivation of virus-containing materials using aminoethylethyleneimine (AEEI), which can significantly reduce labor and energy costs for the manufacture of vaccines and improve the quality of antigenic materials.

An additional technical result from the use of the invention in terms of increasing the antigenic and immunogenic activity of the target product is also achieved through the use of Montanide ISA-70 brand oil adjuvant manufactured by Seppic (France, ISO 9001 standard) in its composition.

The viral isolate, which served as the source for the production strain VGNKI-4 of the PG-3 cattle virus, was isolated from the lungs of 2-3-month-old calves with respiratory pathology. The strain “VGNKI-4” is adapted to the cell culture of the kidneys of the Syrian hamster VNK-21 and to the culture of Taurus-1 calf cells (5).

The strain “VGNKI-4” of the PG-3 cattle virus was deposited on September 30, 1999 with the All-Russian State Collection of microorganism strains 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 ") Under the registration name" VGNKI-4 "DEP.

The strain “VGNKI-4” of the cattle PG-3 virus is characterized by the following features and properties (15).

Morphological features

The strain VGNKI-4 of the cattle PG-3 virus belongs to the family Paramyxoviridae, the genus Parainfluenza, a subtype of Parainfluenza virus 3, and is a classic paramyxovirus in structure. Outwardly, the virus is almost spherical, but polymorphic forms are often found. The floating density in sucrose is 1.197 g / cm ³ . The sedimentation coefficient of virion RNA is 42S. The molecular weight is 4.5 × 10 ⁶ D. The size of the spherical forms is from 80 to 300 nm, but most virions are on average from 130 to 140 nm.

The virus PG-3 cattle strain "VGNKI-4" has 7 structural proteins:

1. Phosphoproteins (P, m. 83 kD).

2. HA-neuraminidase glycoprotein (HN, m. 69 kD).

3. The main nucleocapsid protein (NP, mm 66 kD).

4. Glycoprotein (F, m.m. 55 D).

5. Matrix protein (M, m. 38 D).

6. Protein L-polymerase (m. M. 180 D).

7. Cellular active protein A (m. M. 43 D).

In a two-layer lipid-containing membrane (thickness 10-15 nm), 2 different macromolecules are located: glyagoprotein hemagglutinin neuraminidase (HN) and fusion glycoprotein (F). Both protrude 8 nm above the shell of the virion with an interval of 7-8 nm. Hemagglutinin attaches the virion to the receptors of the host cell containing neuraminic (sialic) acid, and neuraminidase destroys the receptors and thereby helps the elution of the virus in the process of budding from the host cell. F-protein is responsible for the penetration of the virus into the cell, fusing with the cell membrane and responsible for hemolysis, which occurs due to the point hydrolysis of the inactive precursor molecule F ₀ . A known relationship between the structure of glycoproteins and the pathogenicity of the virus. In pathogenic strains, both surface glycoproteins in BHK-21 cells are included in virions only in split form. In apatogenic strains, glycoprotein F is incorporated into virions in the form of a precursor F ₀ . Viruses with undigested glycoproteins have very low biological activity, however, if they are treated with trypsin, they exhibit full biological activity. Protein F is responsible for hemolytic activity and virus-induced karyocytosis. Post-translational cleavage of the precursor protein F ₀ into F ₁ and F _{2 is} carried out by cellular enzymes.

Antigenic properties

The strain "VGNKI-4" virus PG-3 cattle agglutinates red blood cells of guinea pigs, rabbits, pigs, cows, mice, sheep, goats, humans and white mice. Cells infected with cattle PG-3 virus from the VGNKI-4 strain sorb red blood cells of guinea pig, cattle, sheep, rabbit and white mouse. During vaccination, the virus induces the formation of specific antibodies on the 8-14th day after revaccination, the antibodies peak at 16-28 days and last up to one year. The duration and intensity of post-vaccination immunity is ensured mainly by IgG and secretory IgA, the production of which is stimulated by an active vaccination process. Vaccinated calves with a titer of humoral antibodies from 6.0 to 10.3 log _{2 are} resistant to infection with the cattle PG-3 virus. Field isolates of the cattle PG-3 virus have close antigenic affinity with the PG-3 cattle virus of the strain “VGNKI-4”.

Biotechnological characteristics

The strain "VGNKI-4" virus PG-3 cattle is intended for the preparation of live and inactivated vaccines against PG-3 cattle. The virus strain "VGNKI-4" PG-3 cattle is reproduced in cell cultures of PEC, PT, MDBK and BHK-21 and accumulates in titers from 6.5 to 8.5 Ig TCD ₅₀ / cm ³ . The strain "VGNKI-4" is stable and harmless.

Resistance to external factors

The PG-3 virus of cattle of the strain "VGNKI-4" is rapidly destroyed by high temperature. The causative agent is sensitive to chloroform, ether, formalin and detergents.

To obtain antigenic material from the strain “VGNKI-4” of the PG-3 cattle virus, the cell culture of BHK-21 or MDBK is used as a sensitive biological system. The resulting virus-containing material is purified from ballast impurities by centrifugation or any other known method.

For inactivation of the PG-3 virus, AEEI is used, which is added to the purified virus-containing suspension to a concentration of 0.1%. Inactivation of the virus is carried out at 36-37 ° C and a pH of 7.0-7.6 for 24 hours. At the end of inactivation, AEEI is neutralized by adding sodium thiosulfate to the antigenic material. For this, a 1M sodium thiosulfate solution is added to the cooled antigen suspension at the rate of 10% of the volume of the used AEEI.

For the manufacture of the emulsion form of the proposed vaccine, avirulent purified antigenic material is used from the strain VGNKI-4 of the PG-3 virus obtained in a MDBK or BHK-21 cell culture with an infectious activity of at least 6.5 Ig TCD ₅₀ / cm ³ , in an amount not less than 10.0 wt.%.

The viral isolate, which served as the source for the production strain “ARRIAH” of the cattle IRT virus, was isolated from the pathological material of calves who died with the symptoms of IRT during the outbreak in the Minevskoye SEC in Nizhny Novgorod Region in 1998. The vaccine strain "ARRIAH" of cattle IRT virus is adapted to the cultures of BHK-21, MDBK and ZKG cells.

The strain "ARRIAH" of the IRT virus of cattle was deposited on March 12, 2001 in the All-Russian State Collection of microorganism strains 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") under registration the name "ARRIAH-DEPT".

The strain "ARRIAH" virus IRT cattle is characterized by the following features and properties (16).

Morphological properties

The strain “ARRIAH” of the cattle IRT virus belongs to the family Herpesviridae, the subfamily Alphaherpesviridae, the genus Varicellavirus, the HPV-1 group, and has morphological characteristics characteristic of this family.

The strain "ARRIAH" includes a linear 2-strand DNA with a molecular weight of 92-102 kD. Round-shaped virions consist of 4 structural components: a nucleoid, a capsid with a diameter of 120-150 nm, surrounded by a lipid-containing membrane, a membrane (tegument) and an outer membrane. The floating density of virions in the CsCl gradient is 1.27-1.29 g / cm ³ .

A toroidal nucleoid contains DNA and is surrounded by an icosahedral capsid 100-110 nm in diameter containing 162 partially hollow capsomeres. Capsid surrounds the core of the virion, consisting of DNA coated with protein. A membrane of globular material surrounds the capsid. As part of the virion, up to 32 proteins with a molecular weight of up to 200 kD are determined.

Virions have a complex four-level (from the center to the periphery) organization: nucleoid, capsid, supercapsid structure and membrane. The outer shell of virions has a typical structure and contains numerous protrusions (spikes) with a length of about 8 nm. Viruses form characteristic intranuclear inclusions. When passing through the inner sheet of the nuclear membrane into the endoplasmic reticulum, the virions are covered with an additional membrane containing glycoproteins and lipids.

Antigenic properties

According to its antigenic properties, the strain “ARRIAH” of the cattle IRT virus belongs to group 1 of cattle herpes viruses. The virus is stably neutralized by homologous antiserum. The virus has hemagglutinating activity (GA activity), reacting with antibodies from sick or immunized animals in an indirect hemagglutination reaction (RNGA). When vaccinating laboratory and naturally susceptible animals, the viral antigen induces the formation of specific antibodies detected in RNGA at a dilution of 1: 16-1: 256.

Biotechnological characteristics

The virus IRT cattle strain "ARRIAH" is reproduced in cell cultures VNK-21, MDBK and ZKG, causing a cytopathic effect (CPD), and accumulates in titers from 6.5 to 8.0 Ig TCD ₅₀ / cm during 48-96 hours of incubation ³ . The results of adaptation of the RTI virus for cattle of the strain ARRIAH to the indicated cell cultures are presented in tables 1, 2 and 3. The strain ARRIAH is stable and retains its properties for 10 passages (observation period).

Genotaxonomic characteristic

Nine structural proteins of the IRT virus strain “ARRIAH” were isolated and characterized: VP 105, VP 90 (hemagglutinin), VP 74, VP 64, VP 54, VP 50, VP 47, VP 40 and VP 31. Glycoproteins g I, g III, g IV are involved in the formation of humoral and cellular immunity. The most effective in this regard is g IV. According to the neutralization reaction, VP 90 and VP 74 have the highest immunogenicity. Protein labeled as VP 8 with m.m. 96 kDa is one of the main proteins of herpes virus-1 cattle. It participates in modulation of the expression of class A genes, stimulates the proliferation of T cells and the formation of antibodies in calves both after infection and after immunization of animals with purified protein. For the induction of virus-neutralizing antibodies to herpesvirus-1 cattle, 4-membered glycoproteins are responsible, the main of which are g 70 and g 97, located in the spines of the virion. In the g IV structure of the IRT virus, 3 neutralizing domains were identified. Domain 1 contains 2 overlapping epitopes, domain 2 contains one epitope. Domain 1 is related to the penetration of the virus into target cells.

Resistance to external factors

The strain ARRIAH of the cattle IRT virus is resistant to low temperatures: at minus 60-70 ° C the virus survives for 7-9 months, at 56 ° C the virus is inactivated after 20 minutes, at 37 ° C - after 4-10 days, at 22 ° C - after 50 days, at 4 ° C the virus activity decreases after 30-40 days only by 1.0 Ig. A formalin solution of 1: 500 inactivates the virus after 24 hours. Acetone, ether, chloroform and ethyl alcohol inactivate it immediately. In an acidic environment, the virus quickly loses its activity, but persists for a long time (up to 9 months) at pH 6-9 and at 4 ° C. Lyophilization has almost no effect on its activity, however, repeated freezing and thawing reduce the virulence and immunogenicity of the virus.

Additional features and properties

Immunogenic activity -100%. Pathogenicity - expressed. Virulence - expressed. Contagiousness - expressed. Oncogenicity is absent.

Free from contamination by bacteria, mycoplasmas and hemagglutinating viruses.

To obtain antigenic material from the ARRIAH strain of cattle IRT virus, monolayer cultures of BHK-21 and MDBK cells grown in 1.5 dm ³ mattresses or rotating vessels of 3 dm ³ are used as a sensitive biological system. If necessary, to obtain antigenic material from the strain "ARRIAH" virus IRT cattle, you can use a cell culture of 3 KG.

The resulting virus-containing material is freed from cellular detritus and other ballast impurities by low-speed centrifugation and the viral raw material is monitored. The production series of cattle IRT virus should have an infectious activity of at least 6.0 Ig TCD ₅₀ / cm ³ and activity in the RDP of at least 4.0 log ₂ .

For inactivation of the IRT virus strain "ARRIAH" use AEEI, which is introduced into the purified virus-containing suspension to a concentration of 0.1%. Inactivation of the virus is carried out at a temperature of 36-37 ° C for 24 hours at a pH of 7.6-7.8 with periodic stirring of the suspension (every 5-6 hours for 3-5 minutes). At the end of inactivation, AEEI is neutralized by adding sodium thiosulfate to the antigenic material. For this, a 1M sodium thiosulfate solution is added to a suspension of antigen cooled to 2-8 ° C at a rate of 10% by volume of the AEEI used. For the manufacture of the emulsion form of the proposed vaccine, antigenic material is used from the ARRIAH strain of cattle IRT virus with activity in the RNGA of at least 4.0 log ₂ in an amount of at least 10.0 wt.%.

The viral isolate, which served as the source for the production strain “ARRIAH” of cattle coronavirus, was isolated from the feces of a newborn calf with impaired gastrointestinal function. The production strain “ARRIAH” of cattle coronavirus is adapted to cell cultures of MDBK, Taurus-1, KST and Vero.

The strain “ARRIAH” of cattle coronavirus was deposited on March 12, 2001 into the All-Russian State Collection of microorganism strains 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”) under the registration name "ARRIAH-DEPT".

The strain “ARRIAH” of cattle coronavirus is characterized by the following features and properties (17).

Morphological features

The strain “ARRIAH” of cattle coronavirus belongs to the family Coronaviridae, the genus Coronavirus, and has morphological characteristics characteristic of the causative agent of coronavirus infection of cattle: a spherical or pleomorphic virion with a diameter of 80-100 nm consists of a nucleocapsid of helical symmetry and a lipoprotein sheath, the surface of which has buobular protrusions with a length of 12-24 nm, forming the likeness of the solar corona. The floating density of the virions in the sucrose gradient is 1.15-1.18 g / cm ³ .

Three structural proteins were found in the virions: asperomeric glycoprotein E2 (180-200 kD), nucleocapsid phosphoprotein N (50-60 kD) and matrix glycoprotein E1 (23-30 kD).

Antigenic properties

The antigen of cattle coronavirus of the strain "ARRIAH" induces the formation of virus-specific antibodies in the body of guinea pigs and rabbits, which are detected in the inhibition of hemagglutination (RTGA). In the blood serum of vaccinated rabbits, the antibody level ranges from 5.4 ± 0.25 to 7.25 ± 0.47 log ₂ (an average of 6.32 ± 0.39 log ₂ ), and in the blood serum of immunized guinea pigs - from 5 , 4 ± 0.38 to 8.2 ± 0.8 log ₂ (on average 6.1 ± 0.49 log ₂ ).

Antibodies in rtga in titers from 7.9 ± 0.35 to 8.9 ± 0.47 log ₂ are detected in blood serum of samples taken 10-15 days before calving, twice vaccinated cows. Timely feeding of such colostrum provides a high level of colostral antibodies (up to 8.0 log ₂ ) in the body of newborn calves.

Biotechnological characteristics

The cattle coronavirus of the ARRIAH strain is reproduced in calf kidney cell cultures (MDBK, Taurus-1), calf coronary vessels (CCT) and in a heterologous monkey kidney cell culture (Vero). At 37 ° C for 3-4 days of incubation, the virus accumulates in an amount of up to 10 ⁸ particles / cm ³ (data of electron microscopic studies). The results of studies on the adaptation of cattle coronavirus strain "ARRIAH" to cell cultures MDBK, Taurus-1, Vero and KST are presented in table 4.

The strain "ARRIAH" of cattle coronavirus has the ability to reproduce in the MDBK cell culture with the same degree of accumulation at 34 ° C. The reproduction period at this temperature increases by almost a day, but allows you to get a double crop of the virus by changing the supporting environment. "Drain" has a high infectious, antigenic and immunogenic activity.

Resistance to external factors

The strain "ARRIAH" coronavirus cattle shows maximum stability in the pH range of 6.0-7.2. Acidic (pH 3.0) and alkaline (pH 11.0) media have an inactivating effect on the virus. The causative agent is resistant to trypsin. The virus is destroyed by fat solvents: chloroform, methylene chloride and trypsin X-100. The inactivated cattle coronavirus antigen of the ARRIAH strain is stable during storage at 4 ° C both in the native state and as part of inactivated vaccines.

Additional features and properties

Antigenic activity has antigenicity in the composition of an inactivated vaccine and drug for hyperimmunization of donors of diagnostic and therapeutic sera.

Reactogenicity - does not possess reactogenic properties.

Stability preserves the initial biological (antigenic) properties when passaged in an MDBK cell culture for 30 passages (observation period).

Pathogenicity - expressed.

Virulence - expressed.

Contagiousness - expressed.

Oncogenicity is absent.

The strain "ARRIAH" cattle coronavirus is free from contamination by bacteria, mycoplasmas and other hemagglutinating viruses.

To obtain antigenic material from the ARRIAH strain of cattle coronavirus, a MDBK cell culture is mainly used as a sensitive biological system.

If necessary, Taurus-1, KST and Vero cell cultures can be used to obtain antigenic material from the ARRIAH strain of cattle coronavirus.

The resulting virus-containing material is purified from ballast impurities by centrifugation or any other known method.

For inactivation of the virus using AAAI, which is added to the purified virus-containing suspension to a concentration of 0.1%. Inactivation of the virus is carried out at 37 ° C for 24 hours. At the end of inactivation, AEEI is neutralized by introducing a 1M sodium thiosulfate solution into the antigen suspension at the rate of 10% of the volume of used AEEI.

For the manufacture of the proposed vaccine, avirulent purified antigenic material from the ARRIAH strain of cattle coronavirus, obtained mainly in the MDBK cell culture with an accumulation degree of at least 10 ⁸ viral particles / cm ³ and activity in the RGA of at least 7.0 log _{2, is} used as an active substance. , in an amount of not less than 10.0 wt.%.

If necessary, the proposed associated vaccine may contain as an active substance an avirulent purified antigenic material from the ARRIAH strain of cattle coronavirus obtained in Taurus-1 cell culture with a degree of accumulation of at least 10 ⁹ viral particles / cm ³ and activity in the RGA of at least 9, 0 log ₂ , in an amount of not less than 10.0 wt.%.

If necessary, the proposed associated vaccine may contain as an active substance an avirulent purified antigenic material from the ARRIAH strain of cattle coronavirus obtained in a culture of KST cells with a degree of accumulation of at least 10 ^7.5 viral particles / cm ³ and activity in the RGA of at least 6, 0 log ₂ , in an amount of not less than 10.0 wt.%.

If necessary, the proposed associated vaccine may contain as an active substance an avirulent purified antigenic material from the ARRIAH strain of cattle coronavirus obtained in Vero cell culture with an accumulation degree of at least 10 ^7.5 viral particles / cm ³ and activity in the RGA of at least 6, 0 log ₂ , in an amount of not less than 10.0 wt.%.

Thus obtained antigenic material from the strain “ARRIAH” of cattle coronavirus is used to manufacture the emulsion form of the proposed associated vaccine against PG-3, IRT and coronavirus infection of cattle.

For the manufacture of the proposed vaccine, the obtained antigenic materials are mixed in a ratio of 1: 1: 1 and the mixture is thoroughly emulsified with an oil adjuvant in a homogenizer in a ratio, wt.%, 30.0-70.0, respectively.

From oil adjuvants it is advisable to use Montanide ISA-70 brand oil adjuvant manufactured by Seppic (France, ISO 9001 standard).

The content of antigens in the proposed vaccine in the above ratio is their effective amount in the drug, ensuring the achievement of a technical result.

The use of the present invention expands the arsenal of highly immunogenic, harmless and areactogenic associated vaccines against PG-3, IRT and coronavirus infection of cattle emulsion inactivated, capable of protecting livestock of various sex and age groups from pathogens PG-3, IRT and coronavirus infection of cattle circulating in Russia.

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

Example 1

For the preparation of the associated vaccine against PG-3, ИРТ and coronavirus infection of cattle emulsion inactivated, production strains of viruses are used: strain “VGNKI-4” of virus PG-3, strain “ARRIAH” of virus IRT and strain “ARRIAH” of coronavirus cattle infection.

The inoculum virus of the production strain “VGNKI-4” of the PG-3 cattle virus obtained in a monolayer culture of MDBK or BHK-21 cells and stored at minus 20 ° C is thawed and frozen twice. The resulting virus-containing liquid infects the suspension cell culture of BHK-21. The multiplicity of infection is 0.1-1.0 TCD ₅₀ / cell. After infection, the cell culture is incubated in glass cultivators with a volume of 6.5 dm ³ and 36.5 dm ³ at 37 ° C for 24-32 hours until more than 70-90% of the cells have a CPD. After that, the resulting virus-containing suspension is once frozen, thawed, poured into a container and samples are taken to control the sterility and infectious activity of the material. Infectious activity of the virus should be at least 6.5 Ig TCD ₅₀ / cm ³ . The resulting virus is purified from ballast impurities by centrifugation. Then the viral suspension is subjected to inactivation with a working solution AEEI. AEEI working solution is prepared 30-60 minutes before it is introduced into the viral suspension by diluting the initial preparation (15-19% aqueous AEEI solution) with sterile demineralized water to a 6% concentration. To avoid changing the pH of the viral suspension, the AEI working solution is previously adjusted with a 5M solution of acetic acid to a pH of 7.8–8.2. Before the introduction of the inactivant, the virus-containing material is heated to 36-37 ° C and the AEEI working solution is added to it to a final concentration of 0.1%. Inactivation of the virus is carried out for 24 hours at 36-37 ° C and pH 7.0-7.6 with periodic stirring (3-5 minutes after 5-6 hours). After inactivation, the antigenic material is cooled to 4-6 ° C and a sample is taken to check for residual virulence. To neutralize the residual AEEI, a 1M sodium thiosulfate solution is added to the antigen suspension at the rate of 10% of the volume of used AEEI.

The resulting antigen is monitored for sterility and antigenic activity. The antigenic activity of the resulting preparation should be at least 6.5 Ig TCD ₅₀ / cm ³ . The resulting antigenic material is used in an amount of not less than 10.0 wt.% For the manufacture of the emulsion form of the proposed vaccine.

A seed virus of the production strain “ARRIAH” of the IRT cattle virus infects a monolayer culture of ZKG or MDBK, or BHK-21 cells grown in 1.5 dm ³ mattresses or in rotating vessels with a volume of 3.0 dm ³ . The dose of infection should be 0.1-0.01 IE / cell. The infected culture is incubated at a temperature of 37 ° C for 48-96 hours until the appearance of the virus CPP in more than 50% of the monolayer cells. The resulting virus-containing suspension is cooled to 4 ° C and, subject to sterility conditions, freed from cell detritus by low-speed centrifugation. The suspension thus purified should be in the form of a clear pink or cherry liquid. The purified suspension is poured into a container from which a sample is taken for the production control of viral raw materials. The production series of cattle IRT virus should have an infectious activity of at least 6.0 Ig TCD ₅₀ / cm ³ and activity in the RDP of at least 4.0 Iog ₂ .

The inactivation of the IRT virus is carried out using a 6% aqueous solution of AEEI, which is introduced into the virus-containing suspension to a final concentration of 0.1-0.2%. AEEI solution is added to the suspension heated to 26-28 ° C with constant stirring. The pH of the suspension is adjusted between 7.6 and 7.8 by the addition of a 5M solution of acetic acid or ammonia. Inactivation of the virus is carried out at a temperature of 36-37 ° C for 24 hours with periodic stirring of the suspension (3-5 minutes every 5-6 hours). At the end of inactivation, the antigenic material is cooled to 2-8 ° C and samples are taken to check for residual infectivity in the MDBK cell culture. To neutralize AEEI, a 1M solution of sodium thiosulfate is added to the suspension of antigenic material at the rate of 10% of the volume of used AEEI. The resulting antigenic material is used in an amount of not less than 10.0 wt.% For the manufacture of the emulsion form of the proposed vaccine.

The seed virus of the production strain "ARRIAH" of cattle coronavirus is stored before use at a temperature of -20 ° C. The seed virus should be free of bacterial and fungal microflora, specific, cause agglutination of red blood cells of mice, have an activity in the RGA of not less than 1: 8 and the concentration of viral particles (according to electron microscopy) in the range from 10 ^5.0 to 10 ^6.5 viral particles / cm ³ (14). Inoculated cattle coronavirus is frozen twice - thawed and used to infect the MDBK cell culture. Before infection, the MDBK cell culture with a high-quality monolayer is washed twice from the growth serum with a Hanks solution containing trypsin (15-20 μg / cm ³ ). The multiplicity of infection is 0.1-1.0 viral particles / cell. After infection, the culture vessels are incubated at 37 ° C for 1.5-2 hours, periodically shaking. Then, the virus-containing material is drained and a support medium containing trypsin (15-20 μg / cm ³ ) is introduced into it. The vessels are incubated at 34 ° C or 37 ° C until the appearance of the virus CPP in more than 90% of the cells. After this, the culture is twice frozen-thawed. The resulting viral material is purified from ballast impurities by low-speed centrifugation at 2500-3000 rpm for 30 minutes. Inactivation of the virus is carried out using AEEI. For this, a 6% AEI working solution, acidified with glacial acetic acid to a pH of 7.8-8.2, is added to the virus-containing suspension. The final concentration of AEEI in the virus-containing suspension should be at least 0.1%. Inactivation of the virus is carried out at a temperature of 36-37 ° C for 24 hours with periodic stirring of the suspension (3-5 minutes every 5-6 hours). The remainder of the AEEI is neutralized by adding a 1M sodium thiosulfate solution at the rate of 10% of the volume of the used AEEI. The resulting antigenic material is examined for sterility, avirulence, the content of viral particles in suspension using an electron microscope and activity in the RGA. For the manufacture of the proposed vaccine, avirulent purified antigenic material from the ARRIAH strain of cattle coronavirus, obtained mainly in the MDBK cell culture with an accumulation degree of at least 10 ⁸ viral particles / cm ³ and activity in the RGA of at least 7.0 log ₂ , in the amount of not less than 10.0 wt.%.

If necessary, the avirulent and purified antigenic material from the ARRIAH strain of cattle coronavirus obtained in Taurus-1 cell culture with a degree of accumulation of at least 10 ⁹ viral particles / cm ³ and activity in the RGA of at least 9.0 log can be used to make the proposed vaccine ₂ , in an amount of not less than 10.0 wt.%.

If necessary, for the manufacture of the proposed vaccine, you can use avirulent and purified antigenic material from the strain "ARRIAH" cattle coronavirus, obtained in the culture of KST cells with a degree of accumulation of at least 10 ^7.5 viral particles / cm ³ and activity in the RGA of at least 6.0 log ₂ , in an amount of not less than 10.0 wt.%.

If necessary, the avirulent and purified antigenic material from the ARRIAH strain of cattle coronavirus obtained in a Vero cell culture with a degree of accumulation of at least 10 ^7.5 viral particles / cm ³ and activity in the RGA of at least 6.0 log can be used to make the proposed vaccine. ₂ , in an amount of not less than 10.0 wt.%.

Thus obtained antigenic materials are used for the manufacture of the proposed vaccine. For this, antigenic materials are mixed in a ratio of 1: 1: 1 and the mixture is thoroughly emulsified with an oil adjuvant in a homogenizer in a ratio, wt.%, 30.0-70.0, respectively.

From oil adjuvants it is advisable to use Montanide ISA-70 brand oil adjuvant manufactured by Seppic (France, ISO 9001 standard).

The proposed vaccine has an optimal component composition, wt.%:

Avirulent purified antigenic material from strain

"VGNKI-4" virus PG-3 cattle obtained in cell culture

animal origin taken from the group consisting of

cell culture MDBK and from cell culture BHK-21, with infectious activity of at least 6.5 Ig TCD ₅₀ / cm ³ - 10.0

Avirulent purified antigenic material from strain

ARRIAH of cattle IRT virus obtained in cell culture

animal origin taken from the group consisting of

VNK-21 cell culture, from MDBK cell culture and from culture

3KG cells, with infectious activity of at least 6.0 lg TCD ₅₀ / cm ³ and activity in the RDP of at least 4.0 log ₂ - 10.0

Avirulent purified antigenic material from strain

ARRIAH of cattle coronavirus obtained in cell culture

animal origin taken from the group consisting of

MDBK cell culture, from Taurus-1 cell culture, from culture

KST cells and from Vero cell culture, in viral concentration

particles providing protective immune activity

antigen in the animal’s body after administration of the target drug to it - 10.0

Oil adjuvant - up to 100.0

The content of antigenic materials in the proposed vaccine within the above ranges is their effective amount in the preparation, ensuring the achievement of a technical result from the use of the invention.

The resulting vaccine is a homogeneous emulsion of white or white-pink color with a slightly viscous consistency. When storing the vaccine, slight exfoliation of mineral oil is allowed on the upper surface of the emulsion and the aqueous phase at the bottom of the vial with a thickness of not more than 1 cm.

The vaccine is monitored for avirulence and sterility, and then packaged in sterile vials.

Sterility of the vaccine is determined in accordance with GOST 28085-89. The virulence of the drug is determined by the method of three consecutive passages of the inactivated antigen in the MDBK cell culture by the absence of CPD.

To test the safety, the vaccine is administered intramuscularly in the thigh area to 10 white mice at a dose of 0.1 cm ³ . The vaccine is considered harmless if white mice remain clinically healthy for 10 days: without any pathological changes. At the injection site, a local tissue reaction is allowed.

The viscosity of the vaccine is determined on a viscometer VPZH-2 and expressed mm ² / sec. The proposed vaccine containing in its composition an oil adjuvant of the brand Montanide ISA-70, forming a type of emulsion "water-oil", has a viscosity of 38.3 ± 0.5 mm ² / sec.

The vaccine is monitored for emulsion stability by centrifugation at 3000 rpm for 30 minutes, as well as during storage at 2-8 ° C and 37 ° C, followed by a visual assessment of the uniformity of the emulsion. The vaccine with the oil adjuvant of the brand Montanide ISA-70 has a high stability of the emulsion during storage under various temperature conditions and during centrifugation.

To test the reactogenicity of the drug, the proposed vaccine is administered intramuscularly in the thigh area to 3 rabbits and 5 guinea pigs at a dose of 2.0 cm ³ and 1.0 cm ^3, respectively. The clinical condition of the animals is monitored for 14 days.

After immunization in rabbits and guinea pigs, a slight local increase in body temperature and the formation of swelling with a diameter of about 1 cm at the injection site were noted. These signs completely disappear by the end of the observation period.

Thus, the proposed vaccine belongs to the type of weakly reactogenic drugs.

Antigenic activity is tested in rabbits. For this, 6 rabbits weighing 2.0-2.5 kg are taken. The vaccine is administered to 4 rabbits subcutaneously in the back region of 2.0 cm ³ . 14 days after the double administration of the vaccine with an interval of 20-25 days, rabbits are taken from the ear vein 2.0-3.0 cm ^{3 of} blood, the blood is kept at a temperature of 37 ° C, the serum is separated and examined for the presence of antibodies to the PG-virus 3 and cattle coronavirus in the hemagglutination inhibition reaction (RTHA), to the cattle IRT virus in the neutralization reaction (pH) or in the indirect hemagglutination reaction (RNGA). Unvaccinated animals (2 heads) are bled simultaneously with vaccinated animals, serum is separated and used for control.

The vaccine series is considered to have passed antigenicity control if the titer of virus-specific antibodies to PG-3 virus and cattle coronavirus in RTHA is not lower than 5.0 log ₂ , and for IRT virus of cattle in pH not lower than 3.0 log ₂ or in RNGA not lower than 4 , 0 log ₂ .

The associated vaccine against PG-3, IRT and coronavirus infection of cattle inactivated emulsion is intended for immunization of cows and calves in farms that are disadvantaged for these diseases. The vaccine is administered to animals intramuscularly in the middle third of the neck in a volume of 2.0 cm ³ twice with an interval of 21-30 days, followed by revaccination after 6 months.

To maintain well-being for these diseases, the following vaccination schedule should be followed:

1) calves are vaccinated, starting from 1-3 days of age twice with an interval of 21-30 days. Revaccination after 6 months;

2) heifers and cows are vaccinated twice with an interval of 21-30 days. Revaccination after 6 months;

3) heifers of a random period are vaccinated twice with an interval of 21-30 days for 1.0-1.5 months before the first mating. Revaccination after 6 months.

Immunity in vaccinated animals is formed 10-15 days after the repeated administration of the vaccine and lasts at least 6 months.

Example 2

The antigenic activity of the associated vaccine against PG-3, ИРТ and coronavirus infection of cattle inactivated inactivated, made as described in example 1, and containing, wt.%:

Avirulent purified antigenic material from strain

"VGNKI-4" virus PG-3 cattle obtained in a cell culture of BHK-21 with infectious activity of 7.0 Ig TCD ₅₀ / cm ³ - 10.0

Avirulent purified antigenic material from strain

ARRIAH of cattle IRT virus obtained in cell culture

VNK-21 with infectious activity of 6.5 Ig TCD ₅₀ / cm ³ and activity in the RDP of 4.0 log ₂ - 10.0

Avirulent purified antigenic material from strain

ARRIAH of cattle coronavirus obtained in cell culture

MDBK with a degree of accumulation of 10 ⁸ viral particles / cm ³ and activity in the RGA of 7.0 log ₂ - 10.0

Oil adjuvant - up to 100.0

The antigenic activity of the obtained preparation was determined on 10 rabbits weighing 2.5-3.0 kg. In the blood serum of vaccinated rabbits, antibodies to cattle coronavirus were detected in rtga at a level of 6.0 ± 0.44 log ₂ , to a cattle erythrocyte virus virus in rnga at a level of 6.2 ± 0.12 log ₂ and to cattle virus PG-3 in rtga at level 6.5 ± 0.3 log ₂ .

The vaccine was also used to immunize 15-25-day-old calves in the farms of the Nizhny Novgorod region. Table 5 shows the results of a study of blood serum of vaccinated young animals. This drug induced the formation of virus-specific antibodies to pathogens PG-3, IRT and coronavirus infection of cattle in titres of 6.73 ± 0.35 log ₂ , 6.43 ± 0.35 log ₂ and 6.68 ± 0.44 log _2, respectively. An increase in antibody level by 1.28-1.95 log ₂ compared with the original. The number of immune animals increased by 17.5-40.0%.

Example 3

The antigenic activity of the associated vaccine against PG-3, ИРТ and coronavirus infection of cattle inactivated by inactivation, made as described in example 1, and containing, wt%:

Avirulent purified antigenic material from strain

"VGNKI-4" virus PG-3 cattle obtained in the culture of cells of BHK-21, with infectious activity of 7.0 Ig TCD ₅₀ / cm ³ - 10.0

Avirulent purified antigenic material from strain

ARRIAH of cattle IRT virus obtained in cell culture

VNK-21 with infectious activity of 6.5 Ig TCD ₅₀ / cm ³ and activity in the RDP of 4.0 log ₂ - 10.0

Avirulent purified antigenic material from strain

ARRIAH of cattle coronavirus obtained in cell culture

MDBK with a degree of accumulation of 10 ⁸ viral particles / cm ³ and activity in the RGA of 7.0 log ₂ - 10.0

Oil adjuvant - up to 100.0

The studies were carried out in ZAO “Peasant Friend” of the Lyskovsky District of the Nizhny Novgorod Region on 5 cows of 4-8 years of age and in ZAO “Name Lakin” of the Sobinsky District of the Vladimir Region on 33 cows of 4-8 years of age. The research results are presented in table 6, which indicate a high antigenic activity obtained in accordance with the invention of the vaccine. The average level of antibodies to cattle PG-3 virus was 6.9 ± 0.62 log ₂ , to cattle IRT virus - 5.8 ± 0.95 log ₂ , to cattle coronavirus 6.3 ± 0.48 log ₂ , which is a sufficient basis for the practical use of the proposed vaccine. When mixing competition of antigens does not occur.

Example 4

The effectiveness of the associated vaccine against PG-3, ИРТ and cattle coronavirus inactivated emulsion inactivated, made as described in example 1, and containing, wt.%:

Avirulent purified antigenic material from strain

"VGNKI-4" virus PG-3 cattle obtained in a cell culture of BHK-21 with infectious activity of 7.0 Ig TCD ₅₀ / cm ³ - 10.0

Avirulent purified antigenic material from strain

ARRIAH of cattle IRT virus obtained in cell culture

VNK-21 with infectious activity of 6.5 Ig TCD ₅₀ / cm ³ and activity in the RDP of 4.0 log ₂ - 10.0

Avirulent purified antigenic material from strain

ARRIAH of cattle coronavirus obtained in cell culture

MDBK with a degree of accumulation of 10 ⁸ viral particles / cm ³ and activity in the RGA of 7.0 log ₂ - 10.0

Oil adjuvant - up to 100.0

Previously, studies of the blood serum of young cattle from dysfunctional respiratory diseases farms were conducted.

The data obtained indicate that antibodies to the PG-3 virus were detected in 52.0% of blood serum samples, to the cattle IRT virus - to 57.9%, to the cattle coronavirus - to 47.9% and to the causative agent of viral diarrhea - to 29.7%.

Antibodies to one virus were found in 11.9% of sera, to two - in 31.3%, to three - in 25.8%, to four - in 19.5%.

The data obtained indicate the circulation of several pathogens in most of the examined households, which indicates the predominance of the associated course of respiratory diseases.

In a series of experiments, blood serum of cattle of different ages was studied. The results obtained indicate that in most animals up to 30-40 days colostral antibodies to these pathogens are detected, and in young animals of older ages - post-infection. Laboratory data are confirmed by the results of a clinical examination, which indicate outbreaks of respiratory diseases 5-10 days after rearrangement of calves at 30-45 days of age.

As a result of studies of paired blood serum samples of young cattle, data were obtained that testify to the participation of cattle coronavirus in the etiopathogenesis of respiratory diseases of young cattle. In the second samples, an increase in titers of antibodies to coronavirus by 1.4-3.6 log ₂ was noted. In the same samples, there was no increase in titers of antibodies to PG-3 and IRT pathogens, or they increased by 0.2-1.5 log ₂ .

Guided by the information received, an epizootological experiment was performed to identify the role of coronavirus in the respiratory pathology of young cattle. For this, in the conditions of maternity wards of five unsafe respiratory diseases households, 10 groups of calves of 15-25 days old with 20-30 heads each were formed.

In each farm, calves of one group were immunized twice with the commercial associated vaccine against PG-3 and RTI of cattle with an emulsion of inactivated emulsion, and animals of the other group were immunized with the proposed associated vaccine against PG-3, IRT and coronavirus infection of cattle.

As a result of a clinical examination conducted 10-20 days after vaccination, cough and rhinitis were noted in 10-20% of the animals vaccinated with the commercial associated vaccine against PG-3 and emulsion inactivated ИРТ. At the same time, in calves vaccinated with the proposed vaccine, clinical signs of respiratory diseases were not observed.

Table 7 shows the results of studies of blood serum from cattle vaccinated with the proposed vaccine. These data indicate the effectiveness of this drug.

Thus, the possibility of improving the herd from respiratory diseases of viral etiology through the use of the associated vaccine against PG-3, IRT and coronavirus infection of cattle has been shown.

Sources of information taken into account when drawing up the description of the invention to the application for the grant of a patent of the Russian Federation for the invention "Associated vaccine against PG-3, IRT and coronavirus infection of cattle emulsion inactivated":

1. Infectious diseases of animals: a Handbook. Ed. D.F. Ozidze. - M .: Agropromizdat, 1987, 288 p.

2. Syurin V.N., Samuilenko A.Ya., Soloviev B.V., Fomina N.V. Viral diseases of animals. - M.: VNIITIBN, 1998, 928 p.

3. Infectious pathology of animals: In 2 T. / Ed. A.Ya. Samuilenko, B.V. Soloviev, E.A. Nepoklonova, E.S. Voronin. - M.: "Academic Book", 2006. - T.1. - 911 p.

4. Pat. RF №2212896, А61К 39/155, C12N 7/00, 09/27/2003

5. Pat. RF №2271220, А61К 39/265, C12N 7/00, А61Р 31/12, 03/10/2006

6. Pat. RF №2301079, А61К 39/215, А61Р 31/12, 06/20/2007

7. Pat. Czechoslovakia No. 249880, A61K 39/295, 39/108, 03/15/1988

8. Pat. Czechoslovakia No. 250849, A61K 39/295, 39/108, 03/15/1988

9. Pat. Czechoslovakia No. 250850, A61K 39/295, 39/108, 03/15/1988 (prototype).

10. Pat. RF №2145236, А61К 39/295, 39/108, 39/215, 39/245, 39/12, C12N 7/00 (C12N 7/00, C12R 1:93), 02/10/2000.

11. WO 2002/062 382, A61K 39/15, 39/215, 39/08, 39/108, 08/15/2002.

12. Guidance on the use of the inactivated combined vaccine against ИРТ, ПГ-3, viral diarrhea, respiratory syncytial, rota- and coronavirus disease of calves KOMBOVAK, No. 13-3-04 / 0703 of 03.20.2003, approved by the Department of Veterinary of the Ministry of Agriculture of the Russian Federation March 20, 2003

13. Pat. RF №2261111, А61К 39/295, 09/27/2005

14. Ponomarev A.P., Molchanova A.I. and Uzyumov V.L. Determination of the concentration of FMD virus particles in suspensions. - Veterinary medicine, 1983, No. 1, 22-24.

15. Pat. RF №2182494, А61К 39/155, 05/20/2002

16. Pat. RF №2221040, C12N 7/00, А61К 39/265 (C12N 7/00, C12R 1:93), 01/10/2004

17. Pat. RF №2268937, C12N 7/00, А61К 39/215, А61Р 31/12, 01/27/2006

18. Mironova L.A., Preobrazhenskaya N.K. et al. Domestic lines of transplantable cells of calf kidneys - a substrate for biotechnology. // Biotechnology, 1998, 5, 25-31.

Table 1 The results of the adaptation of the virus IRT cattle strain "ARRIAH" to the cell culture MDBK Inoculation Material Passage number Infection dose CPP manifestation time (h) The titer of the Ig virus TCD ₅₀ / cm ³ 20% suspension of US Pat. material 1st 5 cm ³ 72-96 5.26 Suspension 1st passage MDBK 2nd 1.0 TCD / cell 48-96 6.0 Suspension 2nd passage MDBK 3rd 1.0 TCD / cell 24-48 7.5 Suspension 3rd passage MDBK 4th 0.1 TCD / cell 24-48 7.3 Suspension 4th passage MDBK 5th 0.1 TCD / cell 18-26 8.0

table 2 The results of the adaptation of the virus IRT cattle strain "ARRIAH" to the cell culture of BHK-21 Inoculation Material Passage number Infection dose CPP manifestation time (h) The titer of the Ig virus TCD ₅₀ / cm ³ Suspension from the 3rd passage MDBK 1st 1.0 TCD / cell 72-96 5.76 Suspension of the 1st passage of VNK-21 2nd 1.0 TCD / cell 48-96 6.0 Suspension of the 2nd passage of VNK-21 3rd 1.0 TCD / cell 48-96 6.5 Suspension of the 3rd passage of VNK-21 4th 0.1 TCD / cell 48-72 6.5 Suspension of the 4th passage of VNK-21 5th 0.1 TCD / cell 24-48 7.0

Table 3 The results of adaptation of the virus IRT cattle strain "ARRIAH" to the cell culture 3KG Inoculation Material Passage number Infection dose CPP manifestation time (h) The titer of the Ig virus TCD ₅₀ / cm ³ Suspension from the 3rd passage MDBK 1st 1.0 TCD / cell 24-48 7.0 Suspension 1st passage ZKG 2nd 0.1 TCD / cell 24-48 7.25 Suspension of the 2nd passage ZKG 3rd 0.1 TCD / cell 18-24 8.0 Suspension 3rd passage ZKG 4th 0.1 TCD / cell 18-24 8.5

Table 4 The sensitivity of transplanted cell cultures to cattle coronavirus strain "ARRIAH" Cell culture The accumulation of viral particles in cm ³ (electron microscopy) The titer in the hemagglutination reaction (RGA) in log ₂ MDBK 10 ^7.0-9.0 7.0-10.0 Taurus 1 10 ^9.0 9.0-10.0 Vero 10 ^7.5 6.0-7.0 KST 10 ^7.5 7.0

Table 5 The study of the antigenic activity of the proposed emulsion inactivated vaccine against PG-3, IRT and coronavirus infection of cattle in 15-25 day old calves No. p / p Characteristic IRT cattle, strain "ARRIAH" PG-3 cattle, strain "VGNKI-4" Coronavirus cattle, strain "ARRIAH" Before vaccination After vaccination Before vaccination After vaccination Before vaccination After vaccination one serum count 10 10 10 10 10 10 including antibody-containing 7 10 8 10 6 10 average antibody level (log ₂ ) 4,5 ± 0,15 6.2 ± 0.24 4.0 ± 0.29 5.8 ± 0.22 4.6 ± 0.28 5.8 ± 0.32 2 serum count 10 10 10 10 10 10 including antibody-containing 6 10 9 10 7 10 average antibody level (log ₂ ) 4.8 ± 0.26 6.2 ± 0.47 5.1 ± 0.33 6.5 ± 0.38 4.9 ± 0.24 6.2 ± 0.34 3 serum count 10 10 10 10 10 10 including antibody-containing 8 10 9 10 8 10 average antibody level (log ₂ ) 5.5 ± 0.34 6.6 ± 0.29 4.8 ± 0.4 7.2 ± 0.58 5.3 ± 0.5 7.4 ± 0.47

Table 6 The study of the antigenic activity of cattle coronavirus strain "ARRIAH" in the composition of the proposed emulsion inactivated vaccine against IRT, PG-3 and coronavirus cattle infection in cows Pathogen Immune status Number of cows The average level of antibodies (log ₂ ) IRT virus of cattle, strain "ARRIAH" before vaccination 33 2.85 ± 0.50 before revaccination 33 4.3 ± 0.73 after vaccination 33 5.8 ± 0.95 Virus PG-3 cattle, strain "VGNKI-4" before vaccination 33 4.05 ± 0.41 before revaccination 33 6.6 ± 0.90 after vaccination 33 6.9 ± 0.62 Coronavirus cattle, strain "ARRIAH" before vaccination 33 3.79 ± 0.50 before revaccination 33 5.62 ± 0.76 after vaccination 33 6.3 ± 0.55

Table 7 The results of studies of blood serum of cattle immunized with the proposed vaccine against PG-3, IRT and coronavirus infection of cattle emulsion inactivated №№ Age of animals at the time of vaccination Indicators Cattle coronavirus Virus PG-3 cattle Cattle RTI virus Before vaccination After vaccination Before vaccination After vaccination Before vaccination After vaccination one 15-25 day old calves serum count 40 40 40 40 40 40 including antibody-containing,% 70 one hundred 82.5 one hundred 70 one hundred average antibody level, log ₂ 4.98 ± 0.4 6.7 ± 0.4 4.8 ± 0.4 6.7 ± 0.4 5.2 ± 0.3 6.4 ± 0.4 2 20-30 day old calves serum count thirty thirty thirty thirty thirty thirty including antibody-containing,% 66.7 one hundred 66.7 one hundred 66.3 one hundred average antibody level, log ₂ 5.3 ± 0.3 7.1 ± 0.5 4.8 ± 0.3 6.9 ± 0.5 4.0 ± 0.2 6.6 ± 0.4 3 cows serum count 33 33 33 33 33 33 including antibody-containing,% 78.8 one hundred 75.8 one hundred 72.7 one hundred average antibody level, log ₂ 3.8 ± 0.5 6.3 ± 0.6 4.1 ± 0.4 6.6 ± 0.9 2.9 ± 0.5 5.8 ± 1.0 four cows serum count 12 12 12 12 12 12 including antibody-containing,% 75 one hundred 66.7 one hundred 88.3 one hundred average antibody level, log ₂ 5.4 ± 0.5 8.7 ± 0.3 5.0 ± 0.6 7.4 ± 0.5 4.6 ± 0.4 5.5 ± 0.4 5 15-25 day old calves serum count fifteen fifteen fifteen fifteen fifteen fifteen including antibody-containing,% 66.7 one hundred 73.3 one hundred 53.3 one hundred average antibody level, log ₂ 5.1 ± 0.3 6.5 ± 0.6 5.0 ± 0.2 7.0 ± 0.4 3.6 ± 0.4 5.4 ± 0.3 6 20-30 day old calves serum count 9 9 9 9 9 9 including antibody-containing,% 55.6 one hundred 77.8 one hundred 66.7 one hundred average antibody level, log ₂ 4.8 ± 0.3 6.9 ± 0.3 4.6 ± 0.5 6.9 ± 0.2 3.6 ± 0.6 5.9 ± 0.4

Claims (15)

1. Associated vaccine against parainfluenza-3 (PG-3), infectious rhinotracheitis (RTI) and coronavirus infection of cattle (cattle) inactivated emulsion containing the active substance and the target additive, characterized in that as the active substance it contains a mixture of avirulent purified antigenic material from strain "VGNKI-4" virus PG-3 cattle, fam. Paramyxoviridae, genus Parainfluenza, subtype Parainfluenza virus 3, collection of FSI “VGNKI” “VGNKI-4” DEPT, from avirulent purified antigenic material from strain “ARRIAH” of the cattle IRT virus, fam. Herpesviridae, genus Varicellavirus, collection of the Federal State Institution “VGNKI” “ARRIAH-DEPT” and from avirulent purified antigenic material from the strain “ARRIAH” of cattle coronavirus, fam. Coronaviridae, a genus of Coronavirus, a collection of FGU VGNKI VNIIZZH-DEP taken in a ratio of 1: 1: 1 and in quantities that ensure the protective immune activity of each antigen in the animal’s body after administration of the target drug to it. 2. The associated vaccine according to claim 1, characterized in that it contains avirulent purified antigenic material from the strain “VGNKI-4” of the cattle PG-3 virus obtained in animal cell culture taken from the group consisting of MDBK calf kidney cell culture and from a cell culture of the Syrian hamster VNK-21 kidney cells, in an effective amount. 3. The associated vaccine according to claim 2, characterized in that it contains an avirulent purified antigenic material from the VGNKI-4 strain of PG-3 cattle virus obtained in an MDBK cell culture with an infectious activity of at least 6.5 lg TCD ₅₀ / cm ³ , in an amount of not less than 10.0 wt.%. 4. The associated vaccine according to claim 2, characterized in that it contains an avirulent purified antigenic material from the VGNKI-4 strain of PG-3 cattle virus obtained in a KNK-21 cell culture with an infectious activity of at least 6.5 lg TCD ₅₀ / cm ³ in an amount of not less than 10.0 wt.%. 5. The associated vaccine according to claim 1, characterized in that it contains an avirulent purified antigenic material from a strain "ARRIAH" of the RTI virus of cattle obtained in a cell culture of animal origin, taken from the group consisting of a cell culture of BHK-21, from a cell culture MDBK and from 3KG goat gonad cell culture 3KG, in an effective amount. 6. The associated vaccine according to claim 5, characterized in that it contains an avirulent purified antigenic material from the ARRIAH strain of cattle IRT virus obtained in a culture of BHK-21 cells with an infectious activity of at least 6.0 lg TCD ₅₀ / cm ³ and activity in the reaction of diffuse precipitation (RDP) is not less than 4.0 log ₂ , in an amount of not less than 10.0 wt.%. 7. The associated vaccine according to claim 5, characterized in that it contains avirulent purified antigenic material from the ARRIAH strain of cattle IRT virus obtained in an MDBK cell culture with an infectious activity of at least 6.0 lg TCD ₅₀ / cm ³ and RDP activity not less than 4.0 log ₂ , in an amount of not less than 10.0 wt.%. 8. The associated vaccine according to claim 5, characterized in that it contains an avirulent purified antigenic material from the ARRIAH strain of cattle IRT virus obtained in a 3KG cell culture with an infectious activity of at least 6.0 lg TCD ₅₀ / cm ³ and activity in RDP not less than 4.0 log ₂ in an amount of not less than 10.0 wt.%. 9. The associated vaccine according to claim 1, characterized in that it contains an avirulent purified antigenic material from the ARRIAH strain of cattle coronavirus obtained in a cell culture of animal origin, taken from the group consisting of MDBK cell culture, Taurus calf kidney cell culture -1, from a CTF calf coronary vessel cell culture and from Vero monkey kidney cell culture, in an effective amount. 10. The associated vaccine according to claim 9, characterized in that it contains avirulent purified antigenic material from the ARRIAH strain of cattle coronavirus obtained in an MDBK cell culture with an accumulation degree of at least 10 ⁸ viral particles / cm ³ and activity in the hemagglutination reaction ( RGA) not less than 7.0 log ₂ , in an amount of not less than 10.0 wt.%. 11. The associated vaccine according to claim 9, characterized in that it contains avirulent purified antigenic material from the ARRIAH strain of cattle coronavirus obtained in Taurus-1 cell culture with a degree of accumulation of at least 10 ⁹ viral particles / cm ³ and activity in the RGA not less than 9.0 log ₂ , in an amount of not less than 10.0 wt.%. 12. The associated vaccine according to claim 9, characterized in that it contains an avirulent purified antigenic material from the ARRIAH strain of cattle coronavirus obtained in a culture of KST cells with a degree of accumulation of at least 10 ^7.5 viral particles / cm ³ and activity in RGA not less than 6.0 log ₂ , in an amount of not less than 10.0 wt.%. 13. The associated vaccine according to claim 9, characterized in that it contains avirulent purified antigenic material from the ARRIAH strain of cattle coronavirus obtained in Vero cell culture with an accumulation degree of at least 10 ^7.5 viral particles / cm ³ and activity in the RGA not less than 6.0 log ₂ , in an amount of not less than 10.0 wt.%. 14. The associated vaccine according to claim 1, characterized in that as the target additive it contains an oil adjuvant in an amount of at least 70.0 wt.%. 15. The associated vaccine according to any one of claims 1 to 14, characterized in that it contains a mixture of avirulent purified antigenic materials from the strain “VGNKI-4” of the virus PG-3, from the strain “ARRIAH” of the IRT virus and from the strain “ARRIAH” of the coronavirus Cattle obtained in cell cultures of animal origin, and oil adjuvant in the ratio, wt.%:
Avirulent Purified Antigenic Material from strain "VGNKI-4" virus PG-3 cattle, obtained in the culture of animal cells origin taken from the group consisting from cell culture MDBK and from cell culture BHK-21, with infectious activity not less than 6.5 lg TCD ₅₀ / cm ³ 10.0 Avirulent Purified Antigenic Material from strain "ARRIAH" virus IRT cattle, obtained in the culture of animal cells origin taken from the group consisting from a cell culture of BHK-21, from a cell culture MDBK and from 3KG cell culture, with infectious activity not less than 6.0 lg TCD ₅₀ / cm ³ and activity in RDP not less than 4.0 log ₂ 10.0 Avirulent Purified Antigenic Material from strain "ARRIAH" cattle coronavirus, obtained in the culture of animal cells origin taken from the group consisting from MDBK cell culture, from cell culture Taurus-1, from KST cell culture and from Vero cell cultures in viral concentration particles providing protective immune antigen activity in the animal after administration him the target drug 10.0 Oil adjuvant Up to 100.0