RU2504400C1 - Inactivated emulsion associated vaccine for cattle parainfluenza-3, infectious rhinotracheitis and viral diarrhoea - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to veterinary virology and biotechnology. The vaccine contains an active substance and a target additive. As the active substance, the vaccine contains a mixture of an avirulent purified antigen material of the strain VGNKI-4 of PI-3 virus of the family Paramyxoviridae, the genus Parainfluenza, the subtype Parainfluenza virus 3, the collection of VGNKI No. VGNKI-4-DEP, of an avirulent purified antigen material of the strain VNIIZZh of cattle IRT virus of the family Herpesviridae, the genus Varicellavirus, of the collection of VGNKI VNIIZZh-DEP and of an avirulent purified antigen material of the strain NADL-VNIIZZh of cattle VD virus of the family Flaviviridae, of genus Pestivirus, of the collection FGU VGNKI NADL-VNIIZZh- DEP. The strains are taken in the relation of 1:1:1 in the amounts to provide the protective immune activity of each antigen in an animal's body after the target preparation is introduced.

EFFECT: vaccine induces the high antibody level in immunised animals; the immunity in the immunised animals is formed 10-15 days after the repeated introduction of the vaccine and preserved for at least 6 months; when the above antigens are combined, a synergetic effect is observed that enables reducing the quantity of antigens in the preparations with the preserved and continuous immunity.

27 cl, 9 tbl, 4 ex

Description

The invention relates to the field of veterinary virology and biotechnology and for the preparation and use of a vaccine for the prevention of respiratory diseases in cattle (cattle), and especially in newborn calves.

Respiratory diseases in calves are widespread and cause significant damage to livestock. In terms of breadth of distribution, mortality, forced slaughter and lack of gain, they prevail over all other diseases. According to many researchers, up to 80-100% of calves in the post-weaning period are susceptible to respiratory diseases (1, 2, 3).

As a rule, respiratory diseases of young cattle occur as mixed infections. In the pathogenesis of respiratory infection of newborn calves, the main role belongs to the association of pathogens, such as parainfluenza-3 virus (PG-3), infectious rhinotracheitis (IRT) and viral diarrhea (VD).

It is known that viruses PG-3, IRT and VD of cattle are pathogens that have a direct effect on the respiratory tract of the animal. Moreover, they have common "target cells" in which they multiply.

Extremely widespread, the tendency to cause long-term persistence, and relative resistance to various factors determine the enzootic course of these diseases in farms with poor living and feeding conditions.

Mixed viral infections or complicated by secondary bacterial microflora are especially difficult and present a serious problem in terms of their diagnosis, treatment and prevention.

Vaccination of animals against these infectious diseases is of major importance in the prevention of epizootics.

In the arsenal of the biological industry around the world there are a large number of drugs designed for the specific prevention of viral and mixed infectious respiratory diseases of cattle and calves. Under certain conditions of use, all vaccines are effective, but differ in dosage, specific antigenic composition, combination of biotypes, method of inactivation of the virus, the adjuvant used and the type of cell culture.

However, the use of currently known drugs is not always accompanied by a positive effect due to the low immunogenic activity and the presence of antigenic and immunobiological differences between vaccine strains of the viruses that make up the vaccines and epizootic strains of pathogens circulating in Russia and the CIS countries.

The strategy for combating the listed cattle diseases in many countries, including Russia, is based primarily on the use of live or inactivated vaccines in the form of mono- or associated drugs (1, 2, 3).

Live vaccines are currently widely tested with good results, but they have several disadvantages. 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. In addition, the use of live vaccines containing uninactivated antigens of the RTI and VD virus of cattle is strictly contraindicated for pregnant cows and newborn calves because of the possibility of homologous recombination of vaccine and field strains of the virus, leading to exacerbation of the infection, abortion and death of individual animals.

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

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 (4).

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-PTOV-8 INRV No. 3760 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 a target additive, an oil adjuvant in effective ratios (5).

Known vaccine associated PG-3 rotavirus and coronavirus infections and ehsherihioza RNC inactivated emulsion comprising as active ingredient a 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 (6).

Known associated vaccine against IRT, PG-3, viral diarrhea, respiratory syncytial infection, rota- and coronavirus diseases of calves "COMBOVAC" inactivated adsorbed, containing as a active substance a mixture of avirulent purified antigenic materials from 6 production virus strains: 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 effective ratios iah (7).

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 RS-89 and RS-71 causative agent of respiratory syncytial infection of cattle obtained in sensitive biological systems, and as a target howl adjuvant sorbent additives in effective ratios GOA (8).

The closest analogue is the TRIVAK (VIEV) vaccine for the prevention of ИРТ, ПГ-3 and viral diarrhea - mucosal disease (VD-BS) of cattle, containing a mixture of attenuated viruses from the Herpes virus bovis strain TK-A (VIEV ) B-2 "with an activity of 4.5-6.5 lg TCD ₅₀ / cm ³ strain Virus diarrhea - mucosal disease bovinum" BK-1 (B-1) N28 "with an activity of 4.0-6.0 lg TCD ₅₀ / cm ³ , strain Paramyxovirus "PTK-45/86" with activity of 5.0-7.0 lg TCD ₅₀ / cm ³ (9).

A common significant drawback of the known associated vaccines, including the prototype vaccine, intended for the prevention of massive infectious respiratory diseases of cattle of various age and sex 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) a low titer of the infectious activity of the viral suspensions included in the vaccines, as a result of which the necessary amount of protective antigens against these diseases is not provided in the vaccine dose;

3) the vaccine strains included in the preparations do not ensure the formation of immunity against different subtypes and main antigenic variants of epizootic strains of pathogens circulating on farms;

4) 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.

The problem to which the present invention is directed is to obtain an inactivated emulsion inactivated PG-3, IRT and VD cattle vaccine that provides effective protection of susceptible animals of various sex and age groups in relation to different subtypes and main antigenic variants of epizootic strains of pathogens circulating on farms on the territory of the Russian Federation.

The technical result from the use of the invention is to expand the arsenal of highly immunogenic, harmless and areactogenic associated vaccines of emulsion inactivated against infectious diseases of cattle, accompanied by damage to the respiratory system, as well as to increase the stability, antigenic and immunogenic activity of the associated vaccine and create intense and prolonged immunity in vaccinated animals .

The specified technical result was achieved by creating a vaccine associated against PG-3, IRT and VD cattle emulsion inactivated, characterized by the following set of features.

The proposed vaccine contains as an active substance 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) of the virus IRT cattle and from the strain “NADL-ARRIAH” ( author’s name) VD virus of cattle obtained in cell cultures of animal origin in amounts that provide protective immune activity in the animal’s body when the target drug is administered to it, and as the target additive, an oil adjuvant in an effective ratio NII. 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. The vaccine associated against PG-3, IRT and VD cattle emulsion inactivated.

2. The active substance in the form of a mixture of avirulent purified antigenic materials from the strain VGNKI-4 of the cattle PG-3 virus, from the strain ARRIAH virus of the IRT cattle virus and from the strain NADL-ARRIAH virus of the virus VD cattle obtained in animal cell cultures in amounts that provide protective immune activity of each antigen in the animal’s body with the introduction of the target drug.

3. Targeted supplements.

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. The vaccine associated against PG-3, IRT and VD cattle emulsion inactivated.

2. The active substance.

3. Targeted supplements.

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 " NADL-ARRIAH "virus VD cattle, in an effective amount.

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

1. Avirulent and purified antigenic material from the strain "VGNKI-4" virus PG-3 cattle, obtained in a transplantable culture of mammalian cells, in an effective amount.

2. Avirulent and purified antigenic material from the strain VGNKI-4 of the PG-3 cattle virus obtained in the transplanted cell culture of the Syrian hamster (BHK-21) in an effective amount.

3. Avirulent and purified antigenic material from the VGNKI-4 strain of PG-3 cattle virus obtained in transplanted VNK-21 cell culture with an infectious activity of at least 6.5 lg TCD ₅₀ / cm ³ and hemagglutinating activity of at least 5.0 log ₂ before inactivation, in an amount of 10.0 wt.%.

4. Avirulent and purified antigenic material from a strain of VGNKI-4 of the PG-3 cattle virus obtained in an inoculated calf kidney cell culture (MDBK) in an effective amount.

5. Avirulent and purified antigenic material from VGNKI-4 strain of PG-3 cattle virus obtained in transplantable MDBK cell culture with an infectious activity of at least 6.5 lg TCD ₅₀ / cm ³ and hemagglutinating activity of at least 5.0 log ₂ before inactivation, in an amount of 10.0 wt.%.

6. Avirulent and purified antigenic material from the strain VGNKI-4 of the PG-3 cattle virus obtained in an inoculated calf kidney cell culture (Taurus-2) in an effective amount.

7. Avirulent and purified antigenic material from the VGNKI-4 strain of PG-3 cattle virus obtained in a transplanted Taurus-2 cell culture with an infectious activity of at least 6.5 lg TCD ₅₀ / cm ³ and hemagglutinating activity of at least 5.0 log ₂ before inactivation, in an amount of 10.0 wt.%.

8. Avirulent and purified antigenic material from the strain "ARRIAH" virus RTI of cattle obtained in transplantable culture of mammalian cells in an effective amount.

9. Avirulent and purified antigenic material from the strain "ARRIAH" virus IRT cattle obtained in transplantable culture of goat gonad cells (Ch-91), in an effective amount.

10. Avirulent and purified antigenic material from the ARRIAH strain of cattle IRT virus obtained in a transplanted cell culture of Ch-91 with an infectious activity of at least 6.0 lg TCD ₅₀ / cm ³ before inactivation, in an amount of 10.0 wt.%.

11. Avirulent and purified antigenic material from the strain "ARRIAH" virus IRT cattle obtained in transplantable cell culture MDBK, in an effective amount.

12. Avirulent and purified antigenic material from the ARRIAH strain of cattle ИРТ virus obtained in transplantable MDBK cell culture with an infectious activity of at least 6.0 lg TCD ₅₀ / cm ³ before inactivation, in the amount of 10.0 wt.%.

13. Avirulent and purified antigenic material from the strain "ARRIAH" virus IRT cattle obtained in a transplanted culture of kidney cells of a calf (RBT), in an effective amount.

14. Avirulent and purified antigenic material from the ARRIAH strain of cattle IRT virus obtained in a transplanted RBT cell culture with an infectious activity of at least 6.0 lg TCD ₅₀ / cm ³ before inactivation, in an amount of 10.0 wt.%.

15. Avirulent and purified antigenic material from the strain "NADL-ARRIAH" virus VD cattle obtained in transplantable culture of mammalian cells in an effective amount.

16. Avirulent and purified antigenic material from the strain "NADL-ARRIAH" virus VD cattle obtained in transplantable cell culture of Taurus-2, in an effective amount.

17. Avirulent and purified antigenic material from the strain "NADL-ARRIAH" virus VD cattle obtained in transplantable cell culture of Taurus-2 with an infectious activity of not less than 6.0 lg TCD ₅₀ / cm ³ and activity in ELISA of not less than 6.65 log ₂ before inactivation, in an amount of 10.0 wt.%.

18. Avirulent and purified antigenic material from the strain "NADL-ARRIAH" virus VD cattle obtained in transplantable RBT cell culture, in an effective amount.

19. Avirulent and purified antigenic material from the strain "NADL-ARRIAH" virus VD cattle obtained in transplantable culture of RBT cells with an infectious activity of at least 6.0 lg TCD ₅₀ / cm ³ and activity in ELISA of at least 6.65 log ₂ up to inactivation, in an amount of 10.0 wt.%.

20. Avirulent and purified antigenic material from the strain NADL-ARRIAH of the VD virus of cattle obtained in the transplanted culture of saiga kidney cells (PS) in an effective amount.

21. Avirulent and purified antigenic material from the strain "NADL-ARRIAH" virus VD cattle obtained in transplantable cell culture PS with an infectious activity of at least 6.0 lg TCD ₅₀ / cm ³ and activity in ELISA of at least 6.65 log ₂ up to inactivation, in an amount of 10.0 wt.%.

22. Of the target additives, an oil adjuvant.

23. Oil adjuvant in an amount of 69.6 wt.%.

24. Of the target additives, additional adjuvant saponin.

25. Saponin in an amount of 0.4 wt.%.

26. Avirulent and purified antigenic material from the VGNKI-4 strain of the PG-3 virus, from the ARRIAH strain of the IRT virus and from the NADL-ARRIAH strain of the VD virus of cattle obtained in transplanted mammalian cell cultures, and target additives oil adjuvant and saponin in the ratio, wt.%:

Avirulent and purified antigenic material from strain "VGNKI-4" virus PG-3 cattle, obtained in transplantable cell culture mammals taken from the group comprising MDBK cell culture, BHK-21 cell culture and Taurus-2 cell culture with infectious activity of at least 6.5 log TCD ₅₀ / cm ³ and hemagglutinating activity not less than 5.0 log ₂ before inactivation 10.0 Avirulent and purified antigenic material from strain "ARRIAH" virus IRT cattle, obtained in transplantable cell culture mammals taken from the group comprising MDBK cell culture, Ch-91 cell culture and RBT cell culture with infectious activity of at least 6.0 lg TCD ₅₀ / cm ³ to inactivation 10.0 Avirulent and purified antigenic material from strain "NADL-ARRIAH" virus VD cattle, obtained in transplantable cell culture mammals taken from the group comprising Taurus-2 cell culture, RBT cell culture and PS cell culture with infectious activity of at least 6.0 lg TCD ₅₀ / cm ³ and activity in ELISA at least 6.65 log ₂ to inactivation 10.0 Oil adjuvant 69.6 Saponin 0.4.

As a result of the studies, the authors found that there is no competition between the antigens that make up the proposed vaccine. 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.

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 cell culture of Taurus-1 calf.

The strain “VGNKI-4” of the PG-3 cattle virus was deposited on September 09, 1999 with the All-Russian State Collection of microorganism strains used in veterinary medicine and animal husbandry at the All-Russian State Research Institute for Control, Standardization and Certification of Veterinary Medicines (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 (10).

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 size of the spherical shapes 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. 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 kD).

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

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

7. Cellular active protein A (m. 43 kD).

In a two-layer lipid-containing membrane (thickness 10-I 5 nm), 2 different macromolecules are located: glycoprotein 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 8–14 days after revaccination, antibodies reach a peak by 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”.

Genotaxonomic characteristic

Paramyxoviruses usually consist of a single molecule of single-stranded, linear, helical RNA with a diameter of 15 nm, having a hollow core inside, the model of which resembles an ear. Its molecular weight is approximately 4.5 × 10 ⁶ , which is approximately 5% of the weight of the viral particle. It contains genetic information for 6 structural proteins. Nucleotide sequencing of the genome of the cattle PG-3 virus showed that it consists of 15156 nucleotides.

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, Taurus-2 and BHK-21 and accumulates in titers from 6.5 to 8.5 lg TCD ₅₀ / cm ³ . The adaptation results of the PG-3 cattle virus of the strain “VGNKI-4” to the cell culture of BHK-21 are presented in table 1. The strain “VGNKI-4” is stable and harmless.

Physical properties

The floating density of the virions in the sucrose gradient is in the range of 1.197 g / cm ³ , the sedimentation coefficient of virion RNA is 42S.

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.

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 - retains the original biological (antigenic) properties when passaged in cell culture MDBK, BHK-21, Taurus-2 for 10 serial passages.

Pathogenicity - not pathogenic for guinea pigs, rabbits and white mice.

Virulence - expressed.

Contagiousness - expressed.

Oncogenicity is absent.

Free from contamination by bacteria, mycoplasmas and extraneous viruses.

To obtain the antigenic material from the strain “VGNKI-4” of the PG-3 cattle virus, cultures of BHK-21, or Taurus-2, or MDBK are mainly 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, aminoethylethyleneimine (AEEI) is used, which is added to the purified virus-containing suspension to a concentration of 0.10%. 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% of the volume of AEEI used.

For the preparation of the emulsion form of the proposed vaccine, avirulent purified antigenic material is used from the VGNKI-4 strain of the PG-3 virus obtained in VNK-21 cell culture, or Taurus-2, or MDBK with an infectious activity of at least 6.5 lg TCD ₅₀ / cm ³ and hemagglutinating activity of at least 5.0 log ₂ before inactivation, in an amount of at least 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. Vaccine strain ARRIAH of the cattle IRT virus is adapted to RBT, MDBK and Ch-91 cell cultures.

The strain “ARRIAH” of the cattle IRT virus was deposited on March 12, 2001 in the All-Russian State Collection of microorganism strains used in veterinary medicine and livestock, the State institution “All-Russian State Research Institute for Control, Standardization and Certification of Veterinary Medicines” (VGNKI) under the registration name “ARRIAH -DEP ".

The strain "ARRIAH" virus RTI of cattle is characterized by the following features and properties (11).

Morphological properties

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

The strain "ARRIAH" includes a linear 2-strand DNA with a molecular weight of 92 ÷ 102 kD. The virions are round in shape, they 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. A toroidal nucleoid contains DNA and is surrounded by an icosahedral capsid with a diameter of 100-5-110 nm, 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 (RIGA). When vaccinating laboratory and naturally susceptible animals, the viral antigen induces the formation of specific antibodies detected in RIGA at a dilution of 1: 16-5-1: 256.

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 cattle herpesvirus-1, 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.

Biotechnological characteristics

The virus IRT cattle strain "ARRIAH" is reproduced in cell cultures MDBK, Ch-91 and RBT, causing a cytopathic effect (CPD), and accumulates in titers from 6.0 to 8.0 lg TCD ₅₀ / cm during 48 ÷ 96 hours of incubation ³ . The results of adaptation of the RTI virus for cattle of the ARRIAH strain to Ch-91 and RBT cell cultures are presented in Tables 2 and 3. The strain ARRIAH is stable and retains its properties for 10 passages (observation period).

Physical properties

The floating density of virions in the CsCl gradient is 1.27 ÷ 1.29 g / cm ³ .

Resistance to external factors

The strain "ARRIAH" of the IRT virus of cattle is resistant to low temperatures: at minus 60 ÷ 70 ° C the virus survives 7 ÷ 9 months, at 56 ° C the virus is inactivated after 20 minutes, at 37 ° C - after 4 ÷ 10 days, at 22 ° С - after 50 days, at 4 ° С the activity of the virus decreases after 30 ÷ 40 days only by 1.0 lg. 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

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 - retains the original biological (antigenic) properties when passaged in the culture of MDBK, Ch-91 and RBT cells for 10 serial passages.

Pathogenicity - not pathogenic for guinea pigs, rabbits and white mice.

Virulence - expressed.

Contagiousness - expressed.

Oncogenicity is absent.

Free from contamination by bacteria, mycoplasmas and extraneous viruses.

To obtain antigenic material from the ARRIAH strain of cattle IRT virus, monolayer cell cultures of MDBK, Ch-91 and RBT grown in 1.5 dm ³ mattresses or rotating vessels of 3 dm ³ are used as a sensitive biological system. The resulting virus-containing material is freed from cellular detritus and other ballast impurities by low-speed centrifugation or any other known method.

For inactivation of the IRT virus strain "ARRIAH" use AEEI, which is introduced into the purified virus-containing suspension to a concentration of 0.10%. 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% of the volume of 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 obtained in a cell culture of Ch-91, or MDBK, or RBT with an infectious activity of at least 6.0 lg TCD ₅₀ / cm ³ before inactivation, in an amount not less than 10.0 wt.%.

The source for the production of the NADL-ARRIAH virus strain VD of cattle was the reference strain NADL of the VD virus of cattle, obtained by the Federal State Institution ARRIAH on August 11, 2004 from the collection of typical cultures of ATCC (USA).

The production strain "NADL-ARRIAH" is obtained by repeated consecutive passages on sensitive biological systems of cultivation.

The strain "NADL-ARRIAH" is adapted to a transplantable culture of mammalian cells selected from the group comprising a monolayer transplantable culture of saiga kidney cells (PS), a monolayer transplantable culture of calf kidney cells (Taurus-2) and a monolayer transplantable culture of calf kidney cells (RBT), It has stable biological properties that allow it to be used as a production seed for the production of antigenic material for inactivated emulsion inactivated VD vaccine.

The strain "NADL-ARRIAH" virus VD cattle was deposited on August 4, 2009 in the All-Russian state collection of strains of microorganisms used in veterinary medicine and animal husbandry of the Federal state institution "All-Russian state center for the quality and standardization of medicines for animals and feed" (FGU "VGNKI") under registration number (reference) - production strain "NADL-ARRIAH" - DEPT of cattle diarrhea virus (12).

Compared with the original strain, the strain "NADL-ARRIAH" has genetic and phenotypic features. Unlike the NADL strain, the NADL-ARRIAH strain is reproduced in transplantable RBT, PS and Taurus-2 cell cultures, has native, infectious, antigenic and immunogenic activity and retains high antigenic and immunogenic activity after inactivation.

The strain "NADL-ARRIAH" virus VD cattle is characterized by the following features and properties (12).

Morphological features

The strain “NADL ARRIAH” of the VD virus of cattle belongs to the family Flaviviridae, the genus Pestivirus, and has morphological characteristics characteristic of the causative agent of VD cattle: a spherical virion with a diameter of 40 ÷ 60 nm consists of an icosahedral symmetric nucleocapsid containing positive single-stranded RNA.

Antigenic properties

According to its antigenic properties, the strain "NADL-ARRIAH" refers to the first genotype.

The virus is stably neutralized by homologous antiserum.

The virus reacts with antibodies from sick or immunized animals in an enzyme-linked immunosorbent assay (ELISA) and microneutralization (PMN). When vaccinating laboratory and naturally susceptible animals, an inactivated virus induces the formation of virus-specific and virus-neutralizing antibodies in guinea pigs in titers from 10.9 to 11.2 log ₂ in ELISA and 7.0 log ₂ in RMN, from 10.8 in rabbits up to 11.4 log ₂ (ELISA) and from 6.0 to 7.1 log ₂ (RMN), in calves and cows - from 10.2 to 10.7 log ₂ (ELISA) and from 7.6 ± 0, 40 to 7.3 ± 0.31 log ₂ (RMN).

Genotaxonomic characteristic

The genome of the strain "NADL-ARRIAH" virus VD cattle presents a positive single-stranded RNA of about 12,500 nucleotides with a single open reading frame. An open reading frame starts at 386 nucleotides. All protein information is contained in a single reading frame.

10 proteins were isolated and characterized: 4 structural and 6 non-structural. Single-stranded RNA is bound to the p14 / C capsid protein surrounded by external glycoproteins (dr25 / E1, dr53 / E2, dr48 / E0), and the lipid membrane.

External glycoprotein gp53 / E2 induces the production of virus-neutralizing antibodies in high titers in animals. Protein dr48 / E0 is able to induce a high level of antibodies in infected animals, but with low virus-neutralizing activity. In infected animals, antibodies to gp25 / E1 are produced in low titers.

By RT-PCR, a portion of the cattle diarrhea virus genome was amplified, including 149 n.a. 5'-untranslated area.

As a result of the studies, it was found that the strain "NADL-ARRIAH" virus VD cattle is closely related to the reference strain "NADL", which differs from one nucleotide in the analyzed area.

Such single substitutions in the protein structure presumably led to significant changes in the antigenic properties of the virus.

Biotechnological characteristic

The strain "NADL-ARRIAH" of the VD virus of cattle is reproduced in transplantable cultures of calf kidney cells (MDBK, Taurus-2, PT, RBT), transplantable cell culture line PS, transplantable cattle intestinal mucosal cell line culture (FBI) and intraspecific hybrid transplantable culture line kidney cells of a pig embryo with pig splenocides (A4C2).

The results of adaptation of the VD virus of cattle of the NADL-ARRIAH strain "to Taurus-2, RBT and PS cell cultures are presented in tables 4, 5, 6. The strain" NADL-ARRIAH "of the virus of VD cattle is stable and retains its properties for 10 passages ( observation period).

Physical properties

The floating density of virions in the sucrose gradient is in the range of 1.12 ÷ 1.13 g / cm ³ , the sedimentation coefficient of virion RNA is 138S.

Resistance to external factors

The strain "NADL-ARRIAH" virus VD cattle is most stable at pH 7.4. The virus is well preserved in its native form at 4 ° C, minus 20 ° C and minus 40 ° C. The virus is sensitive to ether, chloroform, trypsin, acidic pH (3.0) and sodium deoxychalate; at 37 ° C dies after 5 days.

The inactivated VD virus of cattle of the NADL-ARRIAH strain is stable when stored at 4 ° C both in the native state and as part of an inactivated vaccine.

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

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 - retains the original biological (antigenic) properties when passaged in Taurus-2, RBT, PS cell culture for 10 serial passages.

Pathogenicity - not pathogenic for guinea pigs, rabbits and white mice.

Virulence - expressed.

Contagiousness - expressed.

Oncogenicity is absent.

Free from contamination by bacteria, mycoplasmas and extraneous viruses.

To obtain antigenic material from the strain “NADL-ARRIAH” of the VD virus of cattle as a sensitive biological system, monolayer cultures of Taypyc-2, RBT and PS cells grown in 1.5 dm ³ mattresses or rotating vessels of 3 dm ³ are used predominantly. The resulting virus-containing material is freed from cellular detritus and other ballast impurities by low-speed centrifugation or any other known method.

For inactivation of the virus VD strain "NADL-ARRIAH" use AEEI, which is introduced into the purified virus-containing suspension to a concentration of 0.15%. 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% of the volume of AEEI used.

For the manufacture of the emulsion form of the proposed vaccine, antigenic material is used from the strain “NADL-ARRIAH” of the VD virus of cattle obtained in Taurus-2 or RBT or PS cell culture with an infectious activity of at least 6.0 lg TCD ₅₀ / cm ³ and activity in ELISA not less than 6.65 log ₂ before inactivation, in an amount of not less than 10.0 wt.%.

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, IRT and VD of cattle emulsion inactivated, production strains of viruses are used: strain VGNKI-4 of the virus PG-3 cattle, strain "ARRIAH" virus IRT cattle and strain "NADL-ARRIAH" virus VD cattle.

The technological process consists of the following technological steps:

obtaining viral suspensions from strains of PG-3 cattle virus “VGNKI-4”, virus IRT cattle “ARRIAH” and virus VD cattle “NADL-ARRIAH”;

determining the activity of each viral suspension;

inactivation of virus-containing culture fluids of each strain separately;

combining the resulting inactivated suspensions

viruses;

add adjuvant;

mixing all components of the vaccine with subsequent packaging of the finished product.

The step for producing viral suspensions according to the invention is based on a Seed lot system - a system in which successive series of a production strain are obtained from the main seed (Master seed) for a certain number of transfers (passages). In the current production, the production seed strain is prepared from the main seed. The production raw material of the culture virus is produced from the working seed (Working seed) or its next generation - working seed. Moreover, the number of transfers from the main seed should not exceed the values established for each specific microorganism or biological product made on their basis.

The process of obtaining a seed system for each virus involves several stages:

- selection of conditioned cell cultures;

- refreshment of the main seed;

- collection of infectious material;

control of sterility and biological activity of the main seed;

- packaging and storage of the main seed;

- preparation of working seed;

- collection of infectious material;

- control of sterility and biological activity of the working seed;

packaging and storage of working seed.

The seed used to make the vaccine is obtained in transplantable cell cultures.

Inactivated emulsion inactivated culture virus PG-3 cattle grown in roller vessels, culture cattle virus IRT grown in 1.5 dm ³ mattresses or in roller vessels, and culture virus VD are used to make the vaccine associated against PG-3, IRT and VD of cattle emulsion inactivated. Cattle grown in 1.5 dm ³ mattresses or in roll vessels.

The vaccine should contain:

1. Antigenic material from the VGNKI-4 strain of PG-3 cattle virus reproduced in the transplanted cell culture of BHK-21, or Taurus-2, or MDBK with infectious activity of at least 6.5 lg TCD ₅₀ / cm ³ and hemagglutinating activity not lower than 5.0 log _2, in an amount of 10.0 wt.%.

2. Antigenic material from the ARRIAH strain of cattle IRT virus reproduced in the transplanted culture of RBT or Ch-91 or MDBK cells with an infectious activity of at least 6.0 lg TCD ₅₀ / cm ³ in an amount of 10.0 wt.% .

3. Antigenic material from the strain "NADL-ARRIAH" virus VD cattle reproduced in transplantable cell culture of Taurus-2, or RBT or PS with an infectious activity of not less than 6.0 lg TCD ₅₀ / cm ³ and activity in ELISA not less than 6, 65 Iog ₂ , in an amount of 10.0 wt.%.

4. Oil adjuvant in an amount of 69.6 wt.%.

5. Saponin in an amount of 0.4 wt.%.

The contents of mattresses, roller vessels are poured into separate containers and cooled at a temperature of 2 ÷ 8 ° C, freed from cellular detritus in a known manner, using low-speed centrifugation. The suspension freed from detritus should look like a clear pink liquid. Then, a sample is taken from the container for the production control of viral raw materials.

The production series of cattle PG-3 virus should have an infectious activity of at least 6.5 lg TCD ₅₀ / cm ³ ; hemagglutinating activity in the RGA is not lower than 5.0 log ₂ .

The production series of the RTI virus of cattle should have an infectious activity of at least 6.0 lg TCD ₅₀ / cm ³ .

The production series of the VD virus of cattle should have an infectious activity of at least 6.0 lg TCD ₅₀ / cm ³ ; activity in ELISA is not lower than 6.65 log ₂ .

Inactivation of viral raw materials is carried out using a 6% solution of AEEI introduced into the viral suspension of each virus to a final concentration of 0.10 ÷ 0.15 wt.%. Inactivation is carried out at a temperature of 37.0 ° C for 24 hours with periodic stirring (every 5 ÷ 6 hours for 3 ÷ 5 min).

At the end of inactivation, the antigenic material of each virus is cooled to a temperature of 2 ÷ 8 ° C and samples are taken to determine the pH, sterility and completeness of inactivation.

To neutralize AEEI, a 1M sodium thiosulfate solution is added to a suspension of antigenic material at the rate of 10% of the volume of used AEEI.

The obtained antigenic material from the strain VGNKI-4 of the bovine PG-3 virus, the strain ARRIAH virus of the IRT cattle virus and the strain NADL-ARRIAH virus of the VD cattle virus is used to prepare the emulsion form of the vaccine against PG-3, IRT and VD cattle.

For this, the antigenic material is thoroughly emulsified with an oil adjuvant and saponin in the ratio, wt.%: 10.0: 10.0: 10.0: 69.6: 0.4, respectively.

The resulting vaccine is an emulsion of white or white-pink color with a slightly viscous consistency. During storage, slight peeling of the mineral oil in the upper and sealing of the emulsion in the lower part of the bottle is allowed. With thorough agitation, the emulsion acquires a uniform structure.

The vaccine is monitored for sterility, stability, completeness of inactivation (avirulence), safety and antigenic activity, and then packaged in sterile vials.

Sterility of the vaccine is determined in accordance with GOST 28085.

The vaccine is monitored for emulsion stability by centrifugation at 400 g for 10 minutes followed by a visual assessment of the uniformity of the emulsion.

The completeness of the inactivation of the drug is determined by the method of three successive passages of the vaccine in the transplanted cell culture by the absence of CPD, as well as in the RGA (for PG-3 virus).

Determination of vaccine safety is carried out on white mice or on naturally susceptible animals - calves of 2–3 months of age. For this, the drug is administered subcutaneously in the back at a dose of 0.1 cm ^{3 to} ten white mice or intramuscularly in the middle third of the neck, 4 cm ^{3 to} two calves (two vaccination doses).

The vaccine is considered harmless if the calves and laboratory animals remain clinically healthy for 10 days without any pathological changes. At the injection site, a local tissue reaction is allowed.

The antigenicity of the vaccine is tested in rabbits. 7 rabbits weighing 2.0-2.5 kg are taken into the experiment. The vaccine is administered to 5 rabbits subcutaneously in the back at 2 cm ³ . Before vaccination and after 28 days after the double administration of the vaccine with an interval of 20-25 days, the blood is taken from rabbits, kept at a temperature of (37 ± 0.5) ° C, serum is separated and examined for the presence of antibodies to the cattle PG-3 virus in the inhibition reaction hemagglutination (RTHA), to the cattle IRT virus in the neutralization reaction (PH), or in the microneutralization reaction (RMN), or in the indirect hemagglutination reaction (RNGA) and to the cattle VD virus in the ELISA or in the neutralization reaction (PH) or in the reaction microneutralization (RMN). In unvaccinated (control) animals (2 heads), blood is collected and examined simultaneously with vaccinated animals. The vaccine series is considered to have passed the test for antigenic activity if the titer of specific antibodies to the PG-3 virus of cattle in RTHA≥1: 32, to the virus for IRT of cattle in PH≥1: 8, or in RMN≥1: 4, or in PHHA≥1 : 16, to the VD virus of cattle in ELISA ≥1: 50, or in PH≥1: 4, or in РМН≥1: 2.

A series of vaccines associated inactivated emulsion containing, wt.%, Was prepared from the obtained antigenic material of the vaccine associated against PG-3, IRT and VD of cattle

Avirulent and purified antigenic material from strain "VGNKI-4" virus PG-3 cattle, obtained in transplantable cell culture BHK-21 with an infectious activity of 6.5 lgTCD ₅₀ / cm ³ and hemagglutinating activity 5.5 log ₂ to inactivation (hereinafter “Antigenic material”) 10.0 Avirulent and purified antigenic material from strain "ARRIAH" virus IRT cattle, obtained in transplantable cell culture Ch-91 with an infectious activity of 6.5 lg TCD ₅₀ / cm ³ to inactivation (hereinafter “Antigenic material”) 10.0 Avirulent and purified antigenic material from strain "NADL-ARRIAH" virus VD cattle, obtained in transplantable cell culture Taurus-2 with infectious activity 6.5 lg TCD ₅₀ / cm ³ and activity in ELISA 6.65 log ₂ to inactivation (hereinafter “Antigenic material”) 10.0 Oil adjuvant 69.6 Saponin 0.4.

Example 2

Tests of the antigenic activity of the proposed vaccine associated against PG-3, IRT and VD cattle inactivated emulsion, manufactured as described in example 1, and containing, wt.%:

Antigenic material 30,0 Oil adjuvant 69.6 Saponin 0.4.

The antigenic activity of the vaccine associated against PG-3, IRT and VD cattle emulsion inactivated was tested in rabbits, which were injected with a vaccine at a dose of 2.0 cm ² . Animals were immunized twice with an interval of 20-25 days. 28 days after the double administration of the vaccine, blood was collected from animals, serum was separated and examined for antibodies to the IRT virus in the pH reaction, to the PG-3 virus in the RTGA reaction, and to the VD virus in ELISA. The research results are presented in table.7.

From the results shown in Table 7, it can be seen that the average level of antibodies in the blood serum of rabbits inoculated with the inactivated emulsion inactivated PG-3, IRT and VD cattle vaccine, for cattle PG-3 virus was 7.0 log ₂ , virus RTI of cattle 5.7 log ₂ and virus VD cattle 7.5 log ₂ .

Thus, the results obtained in laboratory animals indicate a high antigenic activity of the vaccine associated against PG-3, IRT and VD cattle emulsion inactivated.

Example 3

Tests of the antigenic activity of the proposed vaccine associated against PG-3, IRT and VD cattle inactivated emulsion, manufactured as described in example 1, and containing, wt.%:

Antigenic material 30,0 Oil adjuvant 69.6 Saponin 0.4.

The vaccine was used in farms that were unsuccessful for these infections for the prevention of PG-3, IRT and VD of cattle in young cattle. Vaccination was subject to clinically healthy animals aged 7-14 days, that is, before transferring from the dispensary to the calf. Prior to vaccination, the farm noted mass diseases among 1-3-month-old calves with the following clinical picture: depression, loss of appetite, serous discharge from the nose, cough, pneumonia, diarrhea. The number of young cattle in the farm was 495 goals, the death rate among sick young animals reached 10%. The samples of pathological material revealed the genomes of viruses VD, IRT and PG-3 cattle.

2 groups of calves were formed. The first group (10 goals) the vaccine was administered intramuscularly in the middle third of the neck twice with an interval of 20-25 days at a dose of 2 cm ³ . The second group (4 heads) served as control. The animals were monitored for 35 days. The blood serum of animals was examined for the presence of antibodies to cattle VD virus in ELISA, to cattle IRT virus in RIGA, to cattle PG-3 virus in rtga. Serum samples were taken according to the following scheme: before vaccination, before revaccination on day 21 and after revaccination on day 35. The results are presented in table.8.

From table 8 it is seen that in the experimental group after revaccination on day 35 there was a significant increase in the level of antibodies to the VD virus of cattle to 10.9 ± 0.35 log ₂ , to the virus of IRT cattle to 8.6 ± 0.47 log ₂ to cattle PG-3 virus up to 7.8 ± 0.41 log ₂ . Thus, the increase in antibody titer by day 35 after vaccination against the VD virus of cattle was 3.0 log ₂ , the virus of IRT cattle - 3.6 log ₂ , the virus PG-3 cattle - 3.1 log ₂ . In the control group of animals, the antibody titer by day 21 after the start of the experiment gradually decreased and amounted to 5.6 log ₂ for the VD virus, 3.8 log ₂ for the IRT virus and 3.0 log ₂ for PG-3 cattle.

The use of the vaccine allowed to reduce the death rate of young animals on the farm from 10% to 5%. The disease of individual animals proceeded with a less pronounced clinical picture.

Thus, the inactivated emulsion inactivated vaccine against PG-3, IRT and VD of cattle has high antigenic activity, and antibody titers obtained as a result of double immunization of animals reached a sufficient level to protect the animal from these infections.

Example 4

Tests of the effectiveness of the proposed vaccine associated against PG-3, IRT and VD cattle inactivated emulsion, manufactured as described in example 1, and containing, wt.%:

Antigenic material 30,0 Oil adjuvant 69.6 Saponin 0.4.

In 19 livestock farms, the proposed vaccine was tested. Table 9 shows the results of studies of blood serum from cattle inoculated with the vaccine associated against PG-3, IRT and VD cattle emulsion inactivated. These data indicate the effectiveness of this drug.

Thus, the possibility of improving the herd from respiratory diseases of viral etiology by using the associated vaccine against PG-3, IRT and VD of cattle emulsion inactivated has been shown.

Information sources

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

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

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

4. Pat. Czechoslovakia No. 249880; A61K 39/295, 39/108; 04/16/1987

5. Pat. Czechoslovakia No. 250849; A61K 39/295, 39/108; 05/14/1987

6. Pat. Czechoslovakia No. 250850; A61K 39/295, 39/108; 05/14/1987

7. Instruction for the use of the inactivated combined vaccine against infectious rhinotracheitis, parainfluenza-3, viral diarrhea, respiratory syncytial, rota-, coronavirus diseases of calves Kombovak, No. 13-3-04 / 0703 of March 20, 2003, approved by the Department of Veterinary Medicine on March 20, 2003 .2003 g.

8. Pat. RF №2261111; A61K 39/295, 09/27/2005

9. Pat. RF №2111011; A61K 39/295, May 20, 1998 (prototype).

10. Pat. RF No. 2182494; A61K 39/155, 05.20.2002

11. Pat. RF №2221040; C12N 7/00, A61K 39/265 (C12N 7/00, C12R 1:93), 01/10/2004

12. Pat. RF №2449013, C12N 7/00, А61К 39/12, 04/27/2012

Table 1 The cultivation of the virus PG-3 cattle strain "VGNKI-4" to the cell culture of BHK-21 Cell culture Passage number Virus titer in cell culture (lg TCD ₅₀ / cm>) Hemagglutinin titer (Log ₂ ) one 4.7 - VNK-21 2 6.8 3.0 ÷ 4.0 the hospital. 3 7.5 5,0 four 7.5 4.0 ÷ 5.0 one 7.3 4.0 ÷ 5.0 VNK-21 2 7.0 3.0 ÷ 4.0 suspension. 3 7.5 4.0 ÷ 5.0 four 7.8 4.0 ÷ 5.0

table 2 Adaptation of the IRT virus of cattle of the strain ARRIAH to the Ch-91 cell culture in a stationary way Passage number Infection dose Cultivation Time (h) The virus titer in cell culture (lg TCD ₅₀ / cm ³ ) 1st 1.0 TCD / cell 48 ÷ 72 7.0 2nd 0.1 TCD / cell 48 ÷ 72 6.9 3rd 0.1 TCD / cell 24 ÷ 48 7.1 4th 0.1 TCD / cell 24 ÷ 48 7.2

Table 3 Adaptation of the IRT virus of cattle of the strain ARRIAH to the RBT cell culture Passage number The titer of the IRT virus during stationary cultivation (log TCD ₅₀ / cm ³ ) The titer of the IRT virus during roller cultivation (log TCD ₅₀ / cm ³ ) one 6.3 6.2 2 6.5 6.9 3 6.2 7.5 four 7.0 8.5

Table 4 Adaptation of the VD virus of cattle of the strain "NADL-ARRIAH" to the Taurus-2 cell culture Passage number Cultivation Time (h) The virus titer in cell culture (lg TCD ₅₀ / cm ³ ) ELISA virus titer (log ₂ ) 1st 96 ÷ 120 3.8 4.3 2nd 96 ÷ 120 4,5 5.3 3rd 96 ÷ 120 6.5 6.9 5th 72 ÷ 96 6.8 7.1 7th 72 ÷ 96 6.6 7.0

Table 5 Adaptation of the VD virus of cattle of the strain "NADL-ARRIAH" to the RBT cell culture Passage number Cultivation Time (h) The virus titer in cell culture (lg TCD ₅₀ / cm ³ ) ELISA virus titer (log ₂ ) 1st 96 ÷ 120 6.5 7.0 2nd 96 ÷ 120 6.0 6.9 3rd 72 ÷ 96 6.8 7.1 5th 72 ÷ 96 6.8 7.2 7th 72 ÷ 96 6.4 6.6

Table 6 Adaptation of the VD virus of cattle of the strain "NADL-ARRIAH" to PS cell culture Passage number Cultivation Time (h) The virus titer in cell culture (lg TCD ₅₀ / cm ³ ) ELISA virus titer (log ₂ ) 1st 96 ÷ 120 4.1 4.9 2nd 96 ÷ 120 4,5 5,0 3rd 96 ÷ 120 5.5 5.9 5th 72 ÷ 96 6.8 7.0 7th 72 ÷ 96 6.6 7.0

Table 7 Antigenic activity of the vaccine associated against PG-3, IRT and VD cattle emulsion inactivated for rabbits Series No. The average titer of antibodies (log ₂ ) to viruses PG-3 IRT VD one 6.0 5.2 6.0 2 5.5 5.9 6.6 3 5.7 5,4 6.6 four 6.0 6.5 7.0 5 6.0 6.3 6.9 6 10.5 6.6 8.9 7 9,4 4.0 10.6 average 7.0 5.7 7.5

Table 8 Tests on 1-2-week-old calves of the vaccine associated against PG-3, IRT and VD cattle emulsion inactivated Immune status The average titer of antibodies to viruses PG-3 IRT VD Before vaccination. The percentage of antibody-containing sera (%). The average titer of antibodies (log ₂ ) 28.6 4.7 ± 0.89 64.3 5.0 ± 0.90 7.1 7.9 ± 0.92 Before revaccination. The percentage of antibody-containing sera (%). The average titer of antibodies (log ₂ ) 100 6.5 ± 0.57 100 6.7 ± 0.53 85.7 9.2 ± 0.57 After revaccination. The percentage of antibody-containing sera (%). The average titer of antibodies (log ₂ ) 100 7.8 ± 0.41 100 8.6 ± 0.47 100 10.9 ± 0.35

Claims (27)

1. The vaccine associated against parainfluenza-3 (PG-3), infectious rhinotracheitis (IRT) and viral diarrhea (VD) in cattle is inactivated emulsion containing the active substance and target additives, characterized in that it is an active substance contains a mixture of avirulent purified antigenic material from strain "VGNKI-4" virus PG-3 cattle, this. Paramyxoviridae, genus Parainfluenza, subtype Parainfluenza virus 3, collection VGNKI “VGNKI-4” -DEP, from avirulent purified antigenic material from the strain “ARRIAH” of cattle IRT virus, fam. Herpesviridae, genus Varicellavirus, collection VGNKI "ARRIAH-DEPT" and from avirulent purified antigenic material from the strain "NADL-ARRIAH" virus VD cattle, fam. Flaviviridae, genus Pestivirus, collection of FGU “VGNKI” “NADL-ARRIAH” -DEP, in an effective amount. 2. The vaccine according to claim 1, characterized in that it contains avirulent and purified antigenic material from a strain of VGNKI-4 of the PG-3 cattle virus obtained in a transplantable culture of mammalian cells in an effective amount. 3. The vaccine according to claim 2, characterized in that it contains an avirulent and purified antigenic material from the strain VGNKI-4 of the PG-3 cattle virus obtained in the transplanted cell culture of the Syrian hamster (BHK-21) in an effective amount. 4. The vaccine according to claim 3, characterized in that it contains avirulent and purified antigenic material from the VGNKI-4 strain of the PG-3 cattle virus obtained in a transplanted cell culture of BHK-21 with an infectious activity of at least 6.5 lg TCD ₅₀ / cm ³ and hemagglutinating activity of at least 5.0 log ₂ before inactivation, in an amount of 10.0 wt.%. 5. The vaccine according to claim 2, characterized in that it contains avirulent and purified antigenic material from the strain VGNKI-4 of the PG-3 cattle virus obtained in a transplanted culture of calf kidney cells (MDBK) in an effective amount. 6. The vaccine according to claim 5, characterized in that it contains avirulent and purified antigenic material from the strain "VGNKI-4" virus PG-3 cattle obtained in a transplantable MDBK cell culture with an infectious activity of at least 6.5 lg TCD ₅₀ / cm ³ and hemagglutinating activity of at least 5.0 log ₂ before inactivation, in an amount of 10.0 wt.%. 7. The vaccine according to claim 2, characterized in that it contains avirulent and purified antigenic material from the strain VGNKI-4 of the PG-3 cattle virus obtained in a transplanted culture of calf kidney cells (Taurus-2) in an effective amount. 8. The vaccine according to claim 7, characterized in that it contains avirulent and purified antigenic material from the VGNKI-4 strain of the PG-3 cattle virus obtained in a transplantable Taurus-2 cell culture with an infectious activity of at least 6.5 lg TCD ₅₀ / cm ³ and hemagglutinating activity of at least 5.0 log ₂ before inactivation, in an amount of 10.0 wt.%. 9. The vaccine according to claim 1, characterized in that it contains avirulent and purified antigenic material from the strain "ARRIAH" of the RTI virus of cattle obtained in a transplanted culture of mammalian cells in an effective amount. 10. The vaccine according to claim 9, characterized in that it contains an avirulent and purified antigenic material from the ARRIAH strain of cattle IRT virus obtained in a transplanted culture of goat gonad cells (Ch-91) in an effective amount. 11. The vaccine of claim 10, characterized in that it contains avirulent and purified antigenic material from the strain "ARRIAH" virus RTI of cattle obtained in transplantable cell culture Ch-91 with an infectious activity of at least 6.0 lg TCD ₅₀ / cm ³ before inactivation, in an amount of 10.0 wt.%. 12. The vaccine according to claim 9, characterized in that it contains avirulent and purified antigenic material from the strain "ARRIAH" virus IRT cattle obtained in transplantable cell culture MDBK, in an effective amount. 13. The vaccine according to item 12, characterized in that it contains avirulent and purified antigenic material from the strain "ARRIAH" virus IRT cattle obtained in a transplanted cell culture MDBK with an infectious activity of at least 6.0 lg TCD ₅₀ / cm ³ before inactivation in an amount of 10.0 wt.%. 14. The vaccine according to claim 9, characterized in that it contains an avirulent and purified antigenic material from the strain "ARRIAH" virus IRT cattle obtained in transplantable culture of RBT cells in an effective amount. 15. The vaccine according to 14, characterized in that it contains avirulent and purified antigenic material from the strain "ARRIAH" virus IRT cattle obtained in a transplanted culture of RBT cells with an infectious activity of at least 6.0 lg TCD ₅₀ / cm ³ before inactivation in an amount of 10.0 wt.%. 16. The vaccine according to claim 1, characterized in that it contains avirulent and purified antigenic material from the strain "NADL-ARRIAH" virus VD cattle obtained in a transplantable culture of mammalian cells in an effective amount. 17. The vaccine according to clause 16, characterized in that it contains avirulent and purified antigenic material from the strain "NADL-ARRIAH" virus VD cattle obtained in transplantable cell culture of Taurus-2, in an effective amount. 18. The vaccine according to claim 17, characterized in that it contains avirulent and purified antigenic material from the strain "NADL-ARRIAH" virus VD cattle obtained in transplantable cell culture of Taurus-2 with an infectious activity of not less than 6.0 lg TCD ₅₀ / cm ³ and activity in ELISA not less than 6.65 log ₂ before inactivation, in the amount of 10.0 wt.%. 19. The vaccine according to clause 16, characterized in that it contains avirulent and purified antigenic material from the strain "NADL-ARRIAH" virus VD cattle obtained in transplantable culture of RBT cells in an effective amount. 20. The vaccine according to claim 19, characterized in that it contains avirulent and purified antigenic material from the strain "NADL-ARRIAH" virus VD cattle obtained in a transplanted RBT cell culture with an infectious activity of not less than 6.0 lg TCD ₅₀ / cm ³ and activity in ELISA not less than 6.65 log ₂ before inactivation, in the amount of 10.0 wt.%. 21. The vaccine according to claim 16, characterized in that it contains an avirulent and purified antigenic material from the strain NADL-ARRIAH virus of the VD virus of cattle obtained in transplantable culture of saiga kidney cells (PS) in an effective amount. 22. The vaccine according to item 21, characterized in that it contains avirulent and purified antigenic material from the strain "NADL-ARRIAH" virus VD cattle obtained in transplantable cell culture PS with an infectious activity of at least 6.0 lg TCD ₅₀ / cm ³ and activity in ELISA not less than 6.65 log ₂ before inactivation, in the amount of 10.0 wt.%. 23. The vaccine according to claim 1, characterized in that it contains an oil adjuvant from the target additives. 24. The vaccine according to p. 23, characterized in that it contains an oil adjuvant in an amount of 69.6 wt.%. 25. The vaccine according to claim 1, characterized in that of the target additives, it additionally contains an adjuvant saponin. 26. The vaccine according A.25, characterized in that it contains saponin in an amount of 0.4 wt.%. 27. The vaccine according to any one of claims 1 to 26, characterized in that as an active substance it contains a mixture of avirulent and purified antigenic materials from the strain VGNKI-4 of the PG-3 cattle virus, from the strain "ARRIAH" of the cattle IRT virus and from the strain "NADL-ARRIAH" virus VD cattle obtained in transplantable cultures of mammalian cells, and the target additives are oil adjuvant and saponin in the ratio, wt.%:
Avirulent and purified antigenic material from strain "VGNKI-4" virus PG-3 cattle, obtained in transplantable cell culture mammals taken from the group comprising MDBK cell culture, BHK-21 cell culture and Taurus-2 cell culture with infectious activity of at least 6.5 log TCD ₅₀ / cm ³ and hemagglutinating activity not less than 5.0 log ₂ before inactivation 10.0 Avirulent and purified antigenic material from strain "ARRIAH" virus IRT cattle, obtained in transplantable cell culture mammals taken from the group comprising cell culture MDBK, cell culture Ch-91 and infectious RBT cell culture activity of at least 6.0 lg TCD ₅₀ / cm ³ to inactivation 10.0 Avirulent and purified antigenic material from strain "NADL-ARRIAH" virus VD cattle, obtained in transplantable cell culture mammals taken from the group comprising Taurus-2 cell culture, RBT cell culture and PS cell culture with infectious activity of at least 6.0 lg TCD ₅₀ / cm ³ and activity in ELISA at least 6.65 log ₂ to inactivation 10.0 Oil adjuvant 69.6 Saponin 0.4.