RU2269361C2 - Associated emulsion inactivated vaccine against porcine reproductive and respiratory syndrome (prrs) and porcine parvovirus infection (ppvi) - Google Patents

Abstract

FIELD: veterinary virology and biotechnology.

SUBSTANCE: claimed vaccine contains antigenic material from PRRS virus strain, reproduced in passaged cell culture Marc-145 and inactivated with aminoethylethyleneimine (AEEI), antigenic material from PPVI virus strain reproduced in passaged YPK cell culture and inactivated with AEEI, and oil adjuvant in effective ratio. Vaccine represents white or white-rose emulsion. Vaccine is intramuscularly (behind ear) administered to animal in dose of 2 cm³ independently of age thereof. Immunity in vaccinated animals appears over 21-30 days after vaccination and remains for 6 months or more. Vaccine of present invention induces high levels of antibodies against PRRS and PPVI and provides durable passive immunity in young stocks up to 20-25-days age.

EFFECT: innoxious vaccine useful in animal protection of neonatal age from field viral PRRS and PPVI agents.

14 cl, 8 tbl, 11 ex

Description

The invention relates to the field of veterinary virology and biotechnology and can be used in the development and manufacture of means of specific prophylaxis of the reproductive-respiratory syndrome and parvovirus infection of pigs.

Pig reproductive and respiratory syndrome (PRRS) and porcine parvovirus infection (PVIS) are highly contagious pig diseases characterized by impaired reproductive function in sows, abortion, and the birth of dead or weak piglets with high mortality.

PRRS registered in the late 80s of the last century, first in the farms of the United States and Canada, and then in many other countries. By the mid-90s, it covered almost the whole of Europe. In Russia and some other CIS countries, PRRS has been registered since 1993.

The causative agent of PRRS is an RNA-containing virus belonging to the genus Arterivirus of the family Arteriviridae, first isolated in 1991 and called the "Lelystad virus" (1).

Mass outbreaks of PRRS inflict great economic damage on pig farms. According to various sources, annual productivity in an acute PRRS outbreak is reduced by 5-20%.

The PRRS virus has immunosuppressive properties and this creates the prerequisites for the occurrence of secondary viral or bacterial infections. It has been established that in the majority of pig farms unsuccessful according to PRRS, pathogens of a number of diseases circulate at the same time, including the causative agent of PVIS, also causing disturbances in animal reproduction (2).

The causative agent of PVIS is a DNA-containing virus belonging to the genus Parvovirus of the family Parvoviridae.

The association of porcine parvoviruses (PVA) with a violation of the reproductive function of sows in vivo was first established in 1967 in the UK, but the disease was experimentally reproduced after 10 years.

The disease in sows proceeds, as a rule, without visible symptoms. There is only a violation of reproductive function. The virus crosses the placenta, affects the fruits in 1 half of pregnancy, causing their mummification and death. Clinically, the disease manifests itself as idlers, early, in 1 half of gestation, abortion and premature birth. When a disease occurs in previously successful farms, the birth of live piglets per sow per year is reduced by 50-60%, in stationary dysfunctional farms - by 10-20%.

PVIS is quite widespread in the world and in pig farms in our country.

Currently, the main means of combating PRRS and PVIS is specific prophylaxis, for which live and inactivated vaccines are used, both mono- and associated drugs (3).

The advantage of live vaccines because of their technological effectiveness in production and ease of use is beyond doubt, however, the immune response in pigs is not always stable when administered and in some cases causes complications after vaccination.

In the global industrial pig breeding, for specific prophylaxis of PRRS and PVIS, emulsion inactivated monovaccines made from various strains of pathogens reproduced in sensitive biological systems are widely used (4-13).

The main disadvantages of these vaccines are that they have low antigenic and immunogenic activity and / or are unsafe, since they are made using virulent strains that always create problems when meeting the requirements of veterinary and sanitary safety.

In addition, these PRRS and PVIS vaccines are mono-preparations and contain only one of the two vaccine strains and their sequential independent administration has a more severe stress effect on the organism of the immunized animals and requires twice the amount of labor costs for vaccination.

This drawback is partially eliminated by the creation of associated vaccines containing separately or in combination antigens of PRRS and PVIS of different strains.

Known vaccine associated inactivated against PVIS and leptospirosis, containing Leptospira antigens of serological groups Pomona and Tarassovi, virus-containing material of the strain Parvovirusporaina I-82 with hemagglutinating activity (GA activity) 1: 256-1: 1512 GAU / 0.025 cm ³ , selected in optimal ratios , as an inactivant, formalin or formalin and ethyleneimine dimer, and as an adjuvant, aluminum hydroxide and serum protein (14).

A vaccine associated inactivated against PVIS and Aujeszky's disease is known, containing antigenic materials of homologous pathogens in optimal proportions (15).

A known emulsion inactivated vaccine against swine circovirus infection (CVIS) and PVIS is known, containing antigenic materials from strains of homologous viruses reproduced in sensitive biological systems, an inactivant and an oil adjuvant in an effective ratio (16).

A vaccine associated inactivated against PRRS, PVIS, leptospirosis and erysipelas of pigs containing antigenic materials of homologous pathogens of these infections in optimal proportions is known (17).

The main disadvantages of these associated vaccines are their low antigenic and immunogenic activity.

The closest to the present invention in terms of essential features is an inactivated emulsion vaccine against PVIS, containing antigenic material from strain I-82 of a homologous virus reproduced in a sensitive biological system and subjected to inactivation, and an oil adjuvant in an effective ratio (18).

The vaccine prototype has significant disadvantages:

- it has low antigenic and immunogenic activity and does not provide satisfactory protection against epizootic PVA circulating in the Russian Federation;

- it does not create protection against PRRS;

- as an inactivant, it contains formalin, which causes destructive changes in viral proteins and reduces the immunogenic activity of the vaccine.

In this regard, the problem of creating a vaccine associated emulsion inactivated against PRRS and PVIS, with high antigenic and immunogenic activity and safety, continues to be relevant and is the main direction of research to improve them. The need for the development and production of these vaccines is due to the fact that in nine out of ten pig farms in the Russian Federation that are disadvantaged by PRRS, parvovirus is simultaneously circulating. This is confirmed by the research results of both domestic and foreign scientists.

The objective of the present invention was to develop a highly immunogenic and harmless inactivated emulsion vaccine against PRRS and PVIS based on new production strains of homologous viruses isolated on the territory of the Russian Federation that have high biological, antigenic and immunogenic activity in their native form and after inactivation and suitable for manufacture associated emulsion inactivated drug, creating a tense and prolonged immunity in vaccinated animals against PRRS and PVIS pathogens circulating in the territory of Russia.

The technical result from the use of the present invention is to expand the arsenal of inactivated emulsion vaccines against PRRS and PVIS, which have high immunogenic activity and harmlessness and create intense and long-lasting immunity in vaccinated animals against PRRS and PVIS pathogens circulating in Russia.

The specified technical result was achieved by the creation of the vaccine associated emulsion inactivated against PRRS and PVIS, characterized by the following set of features:

1. Inactivated emulsion inactivated vaccine against PRRS and PVIS.

2. Antigenic material from the PRRS pathogen reproduced in a sensitive biological system in an effective amount.

2.1. As antigenic material from the pathogen PRRS antigenic material from the strain "DB-DEPT" of the PRRS virus.

2.2. Antigenic material from the strain "BD-DEPT" of PRRS virus reproduced in a transplantable cell culture of Marc-145.

2.3. Antigenic material from the strain "BD-DEPT" of the PRRS virus reproduced in a transplanted cell culture of Mags-145 with biological activity of at least 6.0 lg TCD ₅₀ / cm ³ .

3. Inactivant.

3.1. Of the inactivants, aminoethylethyleneimine (AEEI).

3.2. AEEI in the form of a 1% aqueous solution.

3.3. AEEI at a concentration of 0.001-0.05%.

4. Antigenic material from the strain "BD-DEPT" of PRRS virus reproduced in a transplanted Marc-145 cell culture with biological activity of at least 6.0 lg TCD ₅₀ / cm ³ and inactivated AEEI, in the amount of 30.0-32 , 0 wt.%.

5. Antigenic material from the PVIS pathogen reproduced in a sensitive biological system in an effective amount.

5.1. As the antigenic material from the PVIS pathogen, the antigenic material is from the Vl-94-DEP strain PVA.

5.2. Antigenic material from the Vl-94-DEP strain of PVA reproduced in the transplanted culture of cells of the PPK piglet kidney.

5.3. Antigenic material from the Vl-94-DEP strain of PVA reproduced in an inoculated culture of cells of the PPK piglet kidney with biological activity of at least 7.5 lg TCD ₅₀ / cm ³ .

5.4. Antigenic material from the VL-94-DEP strain of PVA reproduced in an inoculated cell culture of PPC with GA activity of at least 1: 2048 GAE.

6. Inactivant.

6.1. Of inactivants AEI.

6.2. AEEI in the form of a 1% aqueous solution.

6.3. AEEI at a concentration of 0.05-0.08%.

7. Antigenic material from the Vl-94-DEP strain of PVA reproduced in a transplantable culture of PPC cells with biological activity of at least 7.5 lg TCD ₅₀ / cm ³ and HA activity of at least 1: 2048 GAE and inactivated AEEI, in the amount of 15.0-17.0 wt.%.

8. Antigenic material from the strain "BD-DEPT" of the PRRS virus and antigenic material from the strain "Vl-94-DEPT" in a ratio of at least 2: 1.

9. Oil adjuvant.

9.1. Of the oil adjuvants, the oil adjuvant ARRIAH (19).

9.2. Oil adjuvant ARRIAH in an amount of at least 53.0 wt.%.

9.3. Of the oil adjuvants, the oil adjuvant is Montanide IZA-70.

9.4. Oil adjuvant "Montanide IZA-70" in an amount of at least 53.0 wt.%.

10. A mixture of antigenic materials from the strain "DB-DEPT" of the PRRS virus reproduced in a transplantable Marc-145 cell culture with biological activity of at least 6.0 lg TCD ₅₀ / cm ³ and inactivated AEEI, and from the strain "Vl- 94-DEP "PVA reproduced in a transplantable cell culture of PPC with biological activity of at least 7.5 lg TCD ₅₀ / cm ³ and HA activity of at least 1: 2048 GAE and inactivated AEEI, combined with an oil adjuvant in a ratio of at least 47:53.

11. Antigenic material from the strain “BD-DEPT” of PRRS virus reproduced in a transplanted Marc-145 cell culture with biological activity of at least 6.0 lg TCD ₅₀ / cm ³ and inactivated AEEI, antigenic material from the strain “VL- 94-DEP "PVA reproduced in a transplantable cell culture of PPC with biological activity of at least 7.5 lg TCD ₅₀ / cm ³ and HA activity of at least 1: 2048 GAE and inactivated AEI, and an oil adjuvant in ratio, wt.%:

Antigenic material from the strain "BD-DEP" of PRRS virus 30.0-32.0 Antigenic material from the strain Vl-94-DEP PVS 15.0-17.0 Oil adjuvant Up to 100.0

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

1. Inactivated emulsion inactivated vaccine against PRRS and PVIS.

2. Antigenic material from the strain "BD-DEPT" of the PRRS virus, reproduced in a sensitive biological system and subjected to inactivation, in an effective amount.

3. Antigenic material from the Vl-94-DEP strain PVIS, reproduced in a sensitive biological system and subjected to inactivation, in an effective amount.

4. Oil adjuvant.

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

1. Antigenic material from the strain "BD-DEPT" of the PRRS virus, reproduced in transplantable cell culture Marc-145.

2. Antigenic material from the strain "BD-DEPT" of the PRRS virus reproduced in a transplantable Marc-145 cell culture with biological activity of at least 6.0 lg TCD ₅₀ / cm ³ .

3. Antigenic material from the strain "BD-DEPT" of the PRRS virus reproduced in a transplanted cell culture of Mags-145 with biological activity of at least 6.0 lg TCD ₅₀ / cm ³ and inactivated AEEI, in the amount of 30.0-32 , 0 wt.%.

4. Antigenic material from the VL-94-DEP strain of PVA reproduced in the transplantable cell culture of PPC.

5. Antigenic material from the Vl-94-DEP strain of PVA reproduced in an inoculated culture of PPC cells with biological activity of at least 7.5 lg TCD ₅₀ / cm ³ .

6. Antigenic material from the VL-94-DEP strain of PVA reproduced in an inoculated cell culture of PPC with GA activity of at least 1: 2048 GAE.

7. Antigenic material from the Vl-94-DEP strain of PVA reproduced in a transplantable culture of PPC cells with biological activity of at least 7.5 lg TCD ₅₀ / cm ³ and HA activity of at least 1: 2048 GAE and inactivated AEEI, in the amount of 15.0-17.0 wt.%.

8. Antigenic material from the strain "BD-DEPT" of the PRRS virus and antigenic material from the strain "Vl-94-DEPT" PVA in a ratio of at least 2: 1.

9. From oil adjuvants oil adjuvant ARRIAH.

10. Oil adjuvant ARRIAH in an amount of at least 53.0 wt.%.

11. Of the oil adjuvants, the oil adjuvant "Montanide IZA-70".

12. Oil adjuvant "Montanide IZA-70" in an amount of at least 53.0 wt.%.

13. Antigenic material from the BD-DEP strain of PRRSV virus reproduced in a transplanted Marc-145 cell culture with biological activity of at least 6.0 lg TCD ₅₀ / cm ³ and inactivated AEEI, antigenic material from the Vl- 94-DEP "PVA reproduced in a transplantable cell culture of PPC with biological activity of at least 7.5 lg TCD ₅₀ / cm ³ and HA activity of at least 1: 2048 GAE and inactivated AEI, and an oil adjuvant in ratio, wt.%:

Antigenic material from strain "DB-DEPT" of PRRS virus 30.0-32.0 Antigenic material from the strain "Vl-94-DEP" PVA 15.0-17.0 Oil adjuvant Up to 100.0

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. Inactivated emulsion vaccine against PVIS.

2. Antigenic material from the PVIS pathogen reproduced in a sensitive biological system and subjected to inactivation.

3. Oil adjuvant.

Compared with the prototype vaccine, the essential distinguishing features of the proposed vaccine are that, as the antigenic material from the PVIS pathogen, it contains the antigenic material from the Vl-94-DEP strain PVA and additionally the antigenic material from the BD-DEP strain of the PRRS virus 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. Antigenic material from the strain "BD-DEPT" of the PRRS virus, reproduced in transplantable cell culture Marc-145.

2. Antigenic material from the strain "BD-DEPT" of the PRRS virus reproduced in a transplantable Marc-145 cell culture with biological activity of at least 6.0 lg TCD ₅₀ / cm ³ .

3. Antigenic material from the strain "BD-DEPT" of PRRS virus reproduced in a transplanted Marc-145 cell culture with biological activity of at least 6.0 lg TCD ₅₀ / cm ³ and inactivated AEEI, in the amount of 30.0-32 , 0 wt.%.

4. Antigenic material from the VL-94-DEP strain of PVA reproduced in the transplantable cell culture of PPC.

5. Antigenic material from the Vl-94-DEP strain of PVA reproduced in an inoculated culture of PPC cells with biological activity of at least 7.5 lg TCD ₅₀ / cm ³ .

6. Antigenic material from the VL-94-DEP strain of PVA reproduced in an inoculated cell culture of PPC with GA activity of at least 1: 2048 GAE.

7. Antigenic material from the Vl-94-DEP strain of PVA reproduced in a transplantable culture of PPC cells with biological activity of at least 7.5 lg TCD ₅₀ / cm ³ and HA activity of at least 1: 2048 GAE and inactivated AEEI, in the amount of 15.0-17.0 wt.%.

8. Antigenic material from the strain "BD-DEPT" of the PRRS virus and antigenic material from the strain "Vl-94-DEPT" PVA in a ratio of at least 2: 1.

9. From oil adjuvants oil adjuvant ARRIAH.

10. Oil adjuvant ARRIAH in an amount of at least 53.0 wt.%.

11. Of the oil adjuvants, the oil adjuvant "Montanide IZA-70".

12. Oil adjuvant "Montanide IZA-70" in an amount of at least 53.0 wt.%.

13. Antigenic material from the BD-DEP strain of PRRSV virus reproduced in a transplanted Marc-145 cell culture with biological activity of at least 6.0 lg TCD ₅₀ / cm ³ and inactivated AEEI, antigenic material from the Vl- 94-DEP "PVA reproduced in a transplantable cell culture of PPC with biological activity of at least 7.5 lg TCD ₅₀ / cm ³ and HA activity of at least 1: 2048 GAE and inactivated AEI, and an oil adjuvant in ratio, wt.%:

Antigenic material from strain "DB-DEPT" of PRRS virus 30.0-32.0 Antigenic material from the strain "Vl-94-DEP" PVA 15.0-17.0 Oil adjuvant Up to 100.0

The proposed vaccine expands the arsenal of means of specific prevention of PRRS and PVIS.

The achievement of the technical result from the use of the invention is explained by the fact that the composition of the associated emulsion inactivated vaccine against PRRS and PVIS introduced antigenic materials from new strains of infectious agents: antigenic material from the strain "DB-DEP" PRRS and antigenic material from the strain "Vl-94- DEP "PVA, - with high biological, antigenic and immunogenic activity in their native form and after inactivation and providing a vaccine that creates intense and long-lasting immunity in vaccinated animals.

The authors found that there is no competition between the antigens that are used in the composition of the proposed vaccine.

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

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 oil adjuvant ARRIAH or "Montanid IZA-70" in its composition.

The strain "DB" is a new, previously unknown strain of the PRRS virus, in its native form non-pathogenic.

The initial virus for obtaining the strain “DB” was isolated from the blood serum of pigs with clinical signs of PRRS obtained from the agricultural holding “Dawn” of the Kirov region of the Stavropol Territory in 1996. The production strain "DB" obtained by repeated consecutive passages on a 3-day cell culture Marc-145.

The strain "DB" was deposited in the All-Russian state collection of strains of microorganisms used in veterinary medicine and animal husbandry, the All-Russian State Research Institute of Control, Standardization and Certification of Veterinary Medicines (VGNKI) May 25, 1998 under the registration code "BD-DEP".

The strain "BD-DEPT" is harmless and has a high biological, antigenic and immunogenic activity in its native form and after inactivation. The possibility of its use for the manufacture of vaccine preparations has been experimentally confirmed.

The strain "BD-DEPT" of the PRRSSS virus is characterized by the following features and properties.

Morphological properties

The strain BD-DEP for the PRRS virus belongs to the family Arteriviridae, the genus Arterivirus, is RNA genomic, has morphological characteristics characteristic of the PRRS virus: the shape of the virion is spherical, the size of the virion is 45-75 nm with a core filling 3/4 of the virion with a diameter of 25- 35 nm, surrounded by a lipid membrane.

Antigenic properties

The PRRS virus of the BD-DEP strain is stably neutralized by homologous antiserum. The virus does not have hemagglutinating properties. When vaccinated, the virus induces the formation of specific antibodies detected in an enzyme-linked immunosorbent assay (ELISA). The method of polymerase chain reaction (PCR) revealed genomic RNA of PRRS virus. The nucleotide sequencing of the “DB-DEPT” strain of PRRS virus showed its significant (about 40%) difference from CNCM strain No. 1-1102 and ATCC strain VR-2332, recognized as reference in Europe and North America, respectively. Identification and specificity of PRRS virus was determined in ELISA (IDEXX, USA) with specificity control. The result of the differential diagnosis was negative for Aujeszky's disease virus, swine parvovirus, cattle diarrhea virus and transmissible gastroenteritis virus.

Biotechnological characteristics

The strain "BD-DEPT" shows a high biological, antigenic and immunogenic activity. The strain "BD-DEP" is intended for the preparation of diagnostic and vaccine preparations. The strain "BD-DEPT" of the PRRS virus is reproduced in the cytoplasm of cells of a 3-day-old culture of Marc-145 cells at a temperature of 37 ° C. The original suspension from the blood serum containing the virus was infected with a Marc-145 cell culture. After 7-10 passages within 48-72 hours of incubation, the virus accumulates to a titer of 4.0-4.5 lg TCD ₅₀ / cm ³ .

Virus infectivity was increased by serial fast alternating passages, which reached 7.0-7.5 lg TCD ₅₀ / cm ^{3 by} passage 41. The strain "BD-DEP" is stable and harmless.

Chemo- and genotaxonomic characterization

The strain "DB-DEPT" is an RNA-containing virus. Nucleic acid contains one strand, the surface of the virus is covered with a layer of glycoproteins. The protein shell detects the following fragments:

M - transmembrane protein;

E - membrane protein;

G _L - surface glycoprotein;

G _S - surface glycoprotein;

N is a nucleocapsid protein.

Physical properties

The mass of the virion is from 49 × 10 ³ Yes to 55 × 10 ³ Yes. The floating density in the gradient of cesium chloride is 1.18-1.2 g / cm ³ .

Resistance to external factors

The strain "BD-DEP" is not resistant to solvents (ether, chloroform) and detergents, sensitive to formaldehyde, ultraviolet radiation, gamma radiation and drying.

Additional features and properties

Immunogenic activity - 14 days after the immunization of pigs it causes them to form specific antibodies.

Reactogenicity is absent.

Pathogenicity is absent.

Virulence is absent.

Oncogenicity is absent.

There is no contagiousness.

The attenuation stability was established during 5 passages on sensitive gilts.

Based on the data obtained, it can be argued that the strain "BD-DEPT" of the PRRS virus in antigenic and immunological spectra is an original, taxonomically new, previously unknown strain of the PRRS virus.

Strain "Vl-94" is a new, previously unknown. The original virus for obtaining the strain “Vl-94” was isolated at the All-Russian Research Institute of Animal Welfare in 1994 during a virological examination of the internal organs of aborted (up to 15 cm long) sows brought from the Vladimirsky pig farm in Vladimir Region. The production strain “Vl-94” PVA was obtained by successive passages on the transplanted culture of cells of the kidney of a piglet “Kazan” (PPC).

The resulting strain was deposited in the All-Russian State Collection of microorganism strains used in veterinary medicine and livestock, the All-Russian State Research Institute of Control, Standardization and Certification of Veterinary Medicines (VGNKI) of the Ministry of Agriculture of the Russian Federation on July 1, 1996 under the registration code "Vl-94-DEP".

The strain "Vl-94-DEP" PVA has high biological, antigenic and immunogenic activity in its native form and after inactivation and is characterized by high productivity in sensitive biological systems of cultivation. The possibility of its use for the preparation of vaccine preparations creating intense and prolonged immunity in vaccinated animals has been experimentally confirmed.

Strain "Vl-94-DEP" PVA is characterized by the following features and properties.

Morphological properties

The strain "Vl-94-DEP" PVA belongs to the family Parvoviridae, genus Parvovirus. Mature virion has cubic symmetry, includes 32 capsomeres, 2 or 3 capsid proteins, the diameter of the virion is about 20 nm, does not contain a shell and lipids, mol. the mass is 5.3 × 10 ⁶ Yes. The viral genome is represented by 1-stranded DNA with mol. mass of 1.4 kDa, i.e. about 26.5% of the mass of the full virion, the proportion of guanine + cytosine is 41 ÷ 53%. The floating density in CsCl of the complete infectious virus, incomplete ("empty") virion and extracted virion DNA is 1.38 ÷ 1.395; 1.30 ÷ 1.315 and 1.724 g / cm ^3, respectively. Sedimentation coefficient 110 ÷ 122 S.

Antigenic properties

The virus contains three polypeptides A, B and C with mol. mass 60 ÷ 90 kDa. All the pig PVA isolates compared were antigenically similar, if not completely identical. Their identity is established in the PH and RTGA. The virus has a pronounced antigenic activity and induces the synthesis of virus-neutralizing, complement-binding and precipitating antibodies, as well as anti-hemagglutinin. A study of the antigenic properties of isolated individual virionic polypeptides of the pathogen showed that the antiserum to each structural protein neutralizes the infectious activity of the full virions and interacts with all three structural proteins of the virus. Each individual protein interacts with virus-neutralizing antibodies obtained for the introduction of a complete virus. Virus-specific antigenic determinants are believed to be present on all PVA virion proteins. The biological half-life of antibodies in piglets depends on their weight, on average it is from 18 to 20 days.

Biotechnological characteristics

The strain "Vl-94-DEP" PVA is well propagated in the transplanted cell culture of PPC and accumulates in the titer of 8.0 lg TCD ₅₀ / cm ³ . The transplanted cell culture SPEV is insensitive to the virus. A feature of the cultivation and accumulation of the virus is its dependence on cell division. The virus propagation curve depends on the number of mitoses. It multiplies well in the dividing cells of the kidney of the pig fetus upon infection 48 hours after planting the culture. The highest concentration of the virus is achieved on day 2-3 of cultivation. CPD is characterized by vacuolization and rounding of cells, diffuse granulation. When studying the reproduction cycle by immunofluorescence, it was found that the antigen is detected in the cell nucleus 6 hours after infection and accumulates in maximum quantities by 18-24 hours. The strain "Vl-94-DEP" PVA is intended for the manufacture of inactivated vaccines against PVIS. The strain "Vl-94-DEP" PVA is genetically stable and retains immunobiological properties for 10 passages (observation period).

Chemo- and genotaxonomic characterization

The genome of the PVA strain Vl-94-DEPT is represented by a 1-helical linear DNA molecule with a mol.m. 1.4 kDa. 1-helical DNA consists of approximately 5 thousand nucleotides and encodes 4 proteins - 3 structural and 1 non-structural. At both ends of the DNA there are non-identical, self-complementary nucleotide sequences (palindromes) forming 2-helix hairpins resistant to S1 nuclease; The 3'-terminal hairpin consists of 115 ÷ 116 nucleotides, the 5'-terminal hairpin consists of 200 ÷ 242 nucleotides. Hairpin structures play an important role in DNA replication. Genes encoding structural proteins are located in the 5'-terminal half, and a gene encoding a non-structural protein is located in the 3'-terminal half of DNA. The virions of the strain "Vl-94-DEPT" contains negative DNA. The virus strain "Vl-94-DEPT" is autonomous (non-defective).

Pathogenic properties

The strain "Vl-94-DEP" PVA is pathogenic for this animal species and apatogenous for hamsters and mice. Symptoms of the disease and pathomorphological changes are usually absent in all infected piglets, but specific antibodies are detected. The virus is isolated from seminal vesicles 7-9 days after infection. From day 12, anti-hemagglutinin is detected in the blood serum of animals. Specific antibodies are also found in piglets in contact with infected animals, which indicates contagiousness of the infection. After intracerebral, oral and intranasal infection, no specific reproducible clinical symptoms were observed in the piglets. However, in such animals it was possible to isolate the virus from the stool, blood and most tissues. Specific antibodies were isolated by day 7. When pigs were infected with the virus from Vl-94-DEPT strain at different stages of pregnancy, transplacental infection of the fetuses was observed.

Hemagglutinating properties

The strain "Vl-94-DEP" PVA agglutinates red blood cells of a guinea pig, chicken, cat, rat, mouse, monkey, human and does not agglutinate red blood cells of a ram, horse, pig, cattle, dog, rabbit, hamster and duck. To determine its GA activity, guinea pig erythrocytes are used, the reaction is carried out at 4 ° C, and physiological saline, pH 7.2, is used as a diluent. GA activity in the RGA was 1: 2048 GAE. The virus is not released from red blood cells at 37-40 ° C for one hour, however, in an alkaline buffer (pH 9.0), it completely elutes from them in 30 minutes at room temperature.

Physical properties

The molecular weight of the virions is 5.5-6.2 MDa, a floating density in the CsCl gradient of 1.39 ÷ 1.42 g / cm ³ , a sedimentation coefficient of 110 ÷ 122 S.

Resistance to external factors

The strain "Vl-94-DEP" PVA is resistant to various physical and chemical factors. It remains infectious at pH 3 ÷ 9 and 37 ° C for 1.5 hours, however, it is completely inactivated at pH 2 under the same conditions. The virus is stable at 56 ° C for 2 days, at 70 ° C for 2 hours, and at 80 ° C it is inactivated in 5 minutes. Does not lose infectivity in the culture fluid with 20% fetal bovine serum at 35 ÷ 37 ° C for 15 weeks. It is stable when treated with chloroform, ether and freon in 10-50% concentration for 10 ÷ 30 minutes at room temperature. The virus is resistant to RNA and DNAase and proteases (papain, chemotrypsin, trypsin, pepsin), is resistant to ultrasound, inactivated by UV rays, formalin, ethyleneimine and its derivatives: β-propiolactone, glutaraldehyde. In rooms, the virus remains active for more than 4 months, in pigsties - up to 4 ÷ 6 months.

Additional features and properties

Free from contamination by bacteria, fungal microflora, mycoplasmas and viruses.

Based on the data obtained, it can be argued that the strain "VL-94-DEPT" according to antigenic and immunological spectra is an original, taxonomically new, previously unknown PVA isolate.

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

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

To verify the conformity of the proposed solution to the patentability condition "inventive step", an additional search for known solutions was carried out to identify features included in the distinctive part of the independent claim. The search results showed that the proposed solution does not follow explicitly for the specialist from the prior art set forth in the corresponding section of the description (no solutions having features matching the distinguishing features of the present invention have been identified), and the effect of the transformations provided for by the essential features of the proposed invention has not been identified to achieve a technical result.

Therefore, the proposed vaccine meets the condition of patentability "inventive step".

The essence of the invention is illustrated by examples of its execution, which do not limit the scope of the invention.

Example 1

Antigenic material from the strain "BD-DEPT" of the PRRS virus for the manufacture of the proposed vaccine is prepared from the mother virus grown in a transplanted culture of Marc-145 cells of 3 days old with a cell concentration of more than 100 thousand cells / cm ³ . Patch virus is considered suitable for the production of viral raw materials if it meets the following requirements: infectivity titer after reproduction in a monolayer of Marc-145 cells 6.5 lg TCD ₅₀ / cm ³ , pH 7.2-7.4 and in the absence of contamination. For the manufacture of 20 liters of culture production virus PRRS take 110-120 mattresses with a cell culture with a capacity of 1.5 DM ³ each. After the growth medium is drained and the FBI or nutrient medium is washed once, 5 cm ^{3 of} virus-containing material of the matrovy series are introduced into the mattresses. Virus adsorption is carried out at 37 ° C for an hour. After that, the needle medium is introduced into the mattresses in a volume of 150-200 cm ³ with the addition of 5% fetal cattle serum with antibiotics (gentamicin at a concentration of 50 μg / cm ³ or its analogues).

Cultivation is carried out at 37 ° C for 48-72 hours. For control, 3 mattresses are left uninfected, in which the medium is replaced. Virus propagation in a Marc-145 cell culture is determined by the nature of the CPD with the formation of clusters of spherical cells rising above the monolayer. There should not be any destructive cell changes in the control mattresses. Mattresses in which cytopathic changes are observed with damage to up to 60% of the cell monolayer (usually after 48-72 hours) are selected and frozen at -20 ° C. The virus for the manufacture of the vaccine is obtained by 2-fold freezing and thawing of infected cells, followed by draining the contents of the mattresses into one container, observing sterile conditions. The resulting virus is freed from cell detritus by centrifugation at 1000 rpm./min for 10 minutes. The viral suspension purified from cellular detritus should be a clear pinkish-cherry liquid. Then, a sample is taken from the container for laboratory production control for infectious activity and sterility.

A production series of PRRS virus is considered suitable if it meets the following requirements: an infectivity titer after reproduction in a monolayer of Marc-145 cells, at least 6.0 lg TCD ₅₀ / cm ³ , pH 7.2-7.4 and in the absence of contamination .

The purified virus-containing suspension is subjected to inactivation. Inactivation of the accumulated viral raw materials is carried out using a 1% aqueous solution of AEI, introduced to a final concentration of 0.001-0.05%. For this, an AEEI solution is added to the suspension heated to 26-28 ° C with constant stirring and the pH value is set at 7.6-7.8 by adding 5% succinic acid solution to it. Inactivation is carried out in a thermostat at a temperature of (37 ± 2) ° C for 20-22 hours with periodic stirring. At the end of inactivation, the suspension is cooled to 4-6 ° C, its pH value is set in the range of 7.6-7.8, and antigen samples are taken to test for sterility, avirulence, and immunogenic activity using methods known to those skilled in the art.

Example 2

The antigenic material from the Vl-94-DEP strain PVA for the manufacture of the proposed vaccine is prepared from a mother virus adapted to the transplantable culture of PPC pig kidney cells grown on the parietal nutrient medium (PSP) and exhibiting infectious activity in the PPC cell culture, at least at least 7.5 lg TCD ₅₀ / cm ³ and a GA activity of at least 1: 2048 GAE. To obtain antigenic material, a culture of PPC cells grown in mattresses with a good monolayer of 2 days of age is used. To obtain 20 liters of a suspension of PVA take 110-120 mattresses with a cell culture with a capacity of 1.5 DM ³ each. After the growth medium is drained, 5-7 cm ³ PVA of matrate seedling are introduced into the mattresses. After 40-60 minutes of incubation at a temperature of (37 ± 2) ° C, 150-200 cm ^{3 of} maintenance medium (without cattle serum) are introduced into the mattresses. Infected mattresses are placed in a cultivation thermostat at a temperature of (37 ± 2) ° C. The cultivation period is from 48 to 72 hours. For control, 3 mattresses are left uninfected, in which the growth medium is changed to 200 cm ^{3 of} maintenance medium without serum. After 24 hours of incubation, infected and control mattresses are microscopied daily. Mattresses in which cytopathic changes are observed with damage to up to 60% of the cell monolayer (usually after 48-72 hours) are selected, thrice frozen at a temperature not exceeding -20 ° C and thawed. Then the virus-containing material is cleaned of cellular detritus. To do this, the contents of the mattresses are poured into one container and the cooled virus-containing suspension, subject to sterile conditions, is freed from cell detritus by centrifugation at 1000 rpm for 10 minutes. The suspension purified from cellular detritus should be in the form of a clear pinkish-cherry liquid. Gentamicin (100 IU) is added to the purified suspension and stirred for 20 minutes. Then, a sample is taken from the container for laboratory production control on the sterility, infectious and GA activity of the virus. The production series of PVA must have an infectious activity of at least 7.5 lg TCD ₅₀ / cm ³ and a GA activity of at least 1: 2048 GAE. The purified virus-containing suspension is subjected to inactivation. Inactivation of the accumulated viral raw materials is carried out using a 1% aqueous solution of AEEI. To do this, the AEEI solution is added to the suspension heated to 26-28 ° C with constant stirring to a final concentration of 0.05-0.08% and the pH value is set at 7.6-7.8 by adding 5% succinic acid to the suspension. Inactivation is carried out in a thermostat at a temperature of (37 ± 2) ° C for 20-22 hours with periodic stirring. At the end of inactivation, the suspension is cooled to 4-6 ° C, a pH value of 7.6-7.8 is established, and samples of antigenic material are taken to test for sterility, avirulence and HA activity, using methods known to those skilled in the art.

Example 3

The associated emulsion inactivated vaccine against PRRS and PVIS is prepared by dispersing a mixture of antigenic materials from the strain BD-DEP of the PRRS virus and the strain Vl-94-DEP of PVA with an oil adjuvant.

For this, antigenic materials obtained as described in examples 1 and 2 are mixed with vigorous stirring in a ratio of 2: 1, i.e. 2 parts of antigenic material of PRRS virus and 1 part of PVA, which corresponds to their content in the proposed vaccine, wt.%:

Antigenic material from strain "DB-DEPT" of PRRS virus 30.0-32.0 Antigenic material from the strain "Vl-94-DEP" PVA 15.0-17.0

Then the resulting mixture is emulsified with an oil adjuvant in the ratio of 47:53. As an oil adjuvant use an oil adjuvant ARRIAH or oil adjuvant "Montanide IZA-70" (Seppic, France), because they are identical in immunostimulating activity.

The finished vaccine is collected in a sterile container, Packed in sterile glass bottles and controlled in accordance with the technical conditions.

The vaccine is an emulsion of a white or pink-white color with a slightly viscous consistency. With long-term storage (over 12 months), a slight peeling of mineral oil over a non-stratified homogeneous emulsion is possible. When shaken, the vaccine acquires a homogeneous homogeneous structure.

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

Antigenic material from the strain "BD-DEP" PRRS virus reproduced in transplantable Marc-145 cell culture with biological activity, at least 6.0 lg TCD ₅₀ / cm ³ and inactivated AEEI 30.0-32.0 Antigenic material from the strain VL-94-DEP PVA, reproduced in transplantable cell culture PPK with biological activity of at least 7.5 lg TCD ₅₀ / cm ³ and HA activity of at least 1: 2048 GAE and inactivated AEEI 15.0-17.0 Oil adjuvant Up to 100.0

The content of antigenic materials in the 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 proposed vaccine is used as a preventive measure in dysfunctional and threatened by PRRS and PVIS farms. Sows, pigs and boars-producers are inoculated twice with an interval of 20-30 days three weeks before mating (insemination). Subsequently, previously immunized sows are inoculated once three weeks before mating (insemination), and boars-producers are revaccinated every 6 months.

Piglets born from sows vaccinated against PRRS and PVIS are immunized twice at 1.5-2 months of age with an interval of 20-30 days. The vaccine is administered intramuscularly behind the ear in a dose of 2 cm ³ regardless of the age of the animals.

Immunity in vaccinated animals occurs on 21-30 days after vaccination and lasts at least 6 months. The vaccine induces a high level of antibodies against PRRS and PVIS, provides reliable passive maternal immunity in young animals up to 20-25 days of age, which allows it to be protected from virulent pathogens of PRRS and PVIS in early life.

Example 4

Tests of the immunogenic activity of the vaccine associated inactivated emulsion inactivated against PRRS and PVIS based on the strains "BD-DEP" and "VL-94-DEPT, respectively, made as described in examples 1, 2 and 3, and containing, wt.%:

Antigenic material from the strain "BD-DEP" PRRS virus reproduced in transplantable Marc-145 cell culture with biological activity 6.0 lg TCD ₅₀ / cm ³ and inactivated AEI 32,0 Antigenic material from the strain VL-94-DEP PVA, reproduced in transplantable cell culture PPK with biological activity of 7.5 lg TCD ₅₀ / cm ³ and GA activity 1: 2048 GAE and inactivated AEEI 15.0 Oil adjuvant Up to 100.0

The immunogenic activity of the vaccine was determined on gilts with a live weight of 30-40 kg of 4-6 weeks of age, free of antibodies to the PRRS virus and PVIS. The vaccine was administered 2.0 cm ³ intramuscularly in the neck. In animals, before vaccination and 34 days after vaccination, blood samples were taken and examined in ELISA (kits from IDEXX, USA) for the presence of specific antibodies. Their assessment was carried out according to the value of S / P against the PRRS virus and the titer of HA activity against PVA.

The research results are shown in table 1.

From table 1 it is seen that in the blood sera of vaccinated animals appeared specific antibodies in high titers. Vaccinated animals did not become ill after challenge with their virulent reference PRRS virus, strain Lelystad.

Example 5

Tests of the immunogenic activity of the vaccine associated inactivated emulsion inactivated against PRRS and PVIS based on the strains "DB-DEPT" and "VL-94-DEPT, respectively, manufactured as described in examples 1, 2 and 3, and containing, wt.%:

Antigenic material from the strain "BD-DEP" PRRS virus reproduced in transplantable cell culture Marc-145 with biological activity of 6.5 lg TCD ₅₀ / cm ³ and inactivated AEEI 30,0 Antigenic material from the strain VL-94-DEP PVA, reproduced in transplantable cell culture PPC with biological activity of 8.0 lg TCD ₅₀ / cm ³ and GA activity 1: 2048 GAE and inactivated AEEI 17.0 Oil adjuvant Up to 100.0

The immunogenic activity of the vaccine was determined on gilts with a live weight of 30-40 kg of 4-6 weeks of age, free of antibodies to the PRRS virus and PVIS. The vaccine was administered 2.0 cm ³ intramuscularly in the neck. In animals, before vaccination and 34 days after vaccination, blood samples were taken and examined in ELISA (IDEXX kits, USA) for the presence of specific antibodies. Their assessment was carried out according to the value of S / P against the PRRS virus and the titer of HA activity against PVA.

The research results are presented in table 2.

The data in table 2 indicate that in the blood serum of animals vaccinated with the vaccine against PRRS and PVIS, a high level of post-vaccination antibodies was detected.

Example 6

Tests of the immunogenic activity of the vaccine associated inactivated emulsion inactivated against PRRS and PVIS based on the strains "DB-DEPT" and "VL-94-DEPT, respectively, manufactured as described in examples 1, 2 and 3, and containing, wt.%:

Antigenic material from the strain "BD-DEP" PRRS virus reproduced in transplantable Marc-145 cell culture with biological activity 7.0 lg TCD ₅₀ / cm ³ and inactivated AEI 30,0 Antigenic material from the strain "Vl-94-DEP" of the PVIS virus reproduced in transplantable cell culture PPC with biological activity of 7.5 lg TCD ₅₀ / cm ³ and GA activity 1: 2048 GAE and inactivated AEEI 17.0 Oil adjuvant Up to 100.0

The immunogenic activity of the vaccine was determined on gilts with a live weight of 30-40 kg of 4-6 weeks of age, free of antibodies to the PRRS virus and PVIS. The vaccine was administered 2.0 cm ³ intramuscularly in the neck. In animals, before vaccination and 34 days after vaccination, blood samples were taken and examined in ELISA (kits from IDEXX, USA) for the presence of specific antibodies. Their assessment was carried out according to the value of S / P against the PRRS virus and the titer of HA activity against PVA.

The research results are presented in table 3.

The results presented in table 3 indicate a high immunogenic activity of the emulsion inactivated vaccine against PRRS and PVIS.

Example 7

To test the immunogenic activity of the proposed vaccine made three prototypes of the drug with different concentrations of antigens and oil adjuvant ARRIAH.

After checking for sterility and harmlessness in mice, each sample was injected intramuscularly into the thigh area for 5 rabbits of 1 cm ³ and three gilts in the neck for 2 cm ³ . The animals were constantly monitored. Blood was collected from animals before and 30 days after vaccination. Observation of vaccinated animals showed that the vaccine is harmless, since no deviations in their condition were noted. Blood serum for the presence of antibodies against the PRRS virus was investigated in RNGA, and against PVA - in rtga. The results of studies of blood serum of vaccinated pigs are shown in table 4.

According to table 4, all animals used in the experiment were seronegative, with the exception of individual rabbits in which anti-PVA antibodies were detected in the blood serum in low titers (≤1: 128). After 30 days, specific antibodies against PRRS and PVIS viruses were detected in the blood serum of all vaccinated animals. So, in gilts, antibodies against the PRRS virus on average in groups ranged from 1: 426 ± 107 to 1: 853 ± 213, and in rabbits, from 1: 448 ± 78 to 1: 512 ± 78. Anti-PVA antibody titers were high and the group average was 1: 1024 ± 0-1: 1365 ± 341 in gilts and 1: 512 ± 0-1: 1024 ± 0 in rabbits. The antigen content from 43% to 47% did not significantly affect the immunogenicity of the vaccine.

The data in table 4 indicate that the prepared samples of the emulsion associated vaccine against PRRS and PVIS are harmless and cause a sufficiently high level of specific antibodies in the blood sera of vaccinated animals. In addition, the results indicate the possibility of using rabbits to assess the immunogenicity of the vaccine.

Example 8

The proposed vaccine was tested for immunogenic activity by controlling infection of immunized animals with virulent PRRS virus, strain Lelystad. In addition, pre-vaccinated and pre-challenge paired sera were tested using a commercial ELISA kit (IDEXX). The generalized results of these experiments are presented in table 5.

According to the data in Table 5, six out of nine vaccinated gilts had a high level of antibodies in the blood against PRRS virus after 35 days. So, in ELISA, their S / P ratio ranged from 0.51 to 0.9, and only three gilts had a dubious result. It should be noted that the commercial ELISA kit (IDEXX) was designed to assess the level of antibodies in sick animals and therefore the S / P value of 0.3 to 0.4 is doubtful. Anti-PVA antibody titers were high and ranged from 1: 512 to 1: 2048. Unvaccinated animals remained seronegative.

The results of the control infection showed that all vaccinated gilts, including those with S / P of 0.28; 0.34; 0.35, do not get sick. In the process of observing them, no clinical manifestations of the disease were noted. The body temperature of the vaccinated animals remained normal.

Three control (unvaccinated) gilts fell ill with a typical clinical picture of PRRS (depression, lack of appetite, conjunctivitis, cough, cyanosis of the skin) and with an increase in body temperature.

Example 9

To study the immunogenicity of the proposed vaccine, as well as determine its harmlessness, experiments were conducted on two seronegative pregnant sows (47-50 days of gestation). The vaccine was administered once in a volume of 2 cm ³ intramuscularly (behind the ear). 42 days after the administration of the vaccine (on 90-93 days of gestation), the sows were intranasally infected with the virulent European PRCC virus, strain Lelistad, in a volume of 10 cm ^{3 of} virus-containing liquid with a titer of 4.0 lg MCC ₅₀ / cm ³ . The animals were clinically observed for 4 months. 37 days after vaccination, blood samples were taken and the sera were examined in ELISA (ARRIAH) for the presence of antibodies against PRRS virus. After vaccination and control infection in pregnant sows, no deviations from the physiological norm were noted. The body temperature of the animals remained within the normative indicators, the appetite remained, the animals actively reacted to the environment. There was no local response to vaccine administration.

On the 37th day after vaccination, specific antibodies to PRRS virus in titers 1: 400 and 1: 200 were detected in the blood serum of animals. These sows brought 16 piglets, of which 14 were healthy and 2 stillborn. The safety among living healthy piglets during the suction period was 100%.

Blood samples were taken from suckling piglets and serum was examined for the presence of antibodies to PRRS virus. Before colostrum, piglets remained seronegative, and 4-7 days after colostrum, high titers of antibodies (1: 400) against PRRS virus were detected in the blood serum of newborn piglets.

Thus, the proposed vaccine against PRRS and PVIS is an immunogenic and harmless drug even for pregnant sows, protecting them from the control of infection with virulent PRRS virus, strain Lelystad.

Example 10

The study of the effectiveness of the proposed vaccine was carried out in a number of successful and dysfunctional according to PRRS and PVIS pig farms.

In order to study the intensity of post-vaccination immunity in sows vaccinated with the associated vaccine against PRRS and PVIS, 5 pig farms were studied. The vaccine was administered according to the instructions for the use of the associated emulsion inactivated vaccine against PRRS and PVIS, i.e. sows, pigs and boars-producers were vaccinated for the first time twice with an interval of 21-25 days 2-3 weeks before insemination. Piglets obtained from vaccinated sows were inoculated at 1.5-2 months of age twice with an interval of 20-30 days. Blood samples were taken from vaccinated animals 2-8 weeks after immunization, i.e. before insemination and in the first half of gestation, in the second half of gestation and immediately after farrowing. In addition, blood was collected from piglets obtained from these sows before they received colostrum and at various times after vaccination. Blood serum from animals was studied in the reactions of RNGA, ELISA (ARRIAH) and rtga. The results of studies of blood serum from vaccinated pigs in successful farms are presented in table 6.

From table 6 it is seen that in pigs of different age and sex groups 55-70 days after vaccination in blood serum revealed a high level of post-vaccination antibodies. There were no particular differences in the intensity of immunity in these groups of animals.

The results of studies of blood serum from dysfunctional farms from sows at various stages of gestation and from piglets who did not take colostrum, are presented in table 7.

As can be seen from table 7, in sows vaccinated with the associated vaccine against PRRS and PVIS, a high titer of antibodies against the causative agents of these infections was detected in blood serum. After farrowing, the level of antibodies decreases slightly, but remains quite high. Thus, the proposed immunization scheme provides immunity against PRRS and PVIS at a high level for the entire period of gestation of sows. The absence of antibodies in the blood serum of "dominant" piglets is a direct indicator of the effectiveness of the vaccination used.

Example 11

The efficacy of the associated emulsion inactivated vaccine against PRRS and PVIS was studied in a number of pig farms unsuccessful according to PRRS and PVIS, in which they carried out epizootological analysis before and after vaccination, taking into account the reduction in the percentage of stillbirth and mortality of piglets. The results of the analysis of the epizootic situation in these farms are presented in table 8.

As can be seen from table 8, regular preventive vaccination of animals significantly reduces the number of sows with reproductive pathology (stillbirth and the birth of non-viable piglets) on farms.

Thus, the associated emulsion inactivated vaccine against PRRS and PVIS is an effective drug for the specific prevention of these diseases, it is harmless and has sufficient immunogenicity. Despite the fact that the vaccine was used in farms unsuccessful according to PRRS and PVIS, where there were a large number of sick animals, it allowed to improve the situation and within 5-24 months the number of sows bringing dead piglets decreased to an average of 2.7%.

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

- vaccine associated emulsion inactivated against PRRS and PVIS, embodying the invention, is intended for use in agriculture, namely in veterinary virology and biotechnology;

- for the proposed invention in the form as described in the independent claim, the possibility of its implementation using the means and methods described in the application or known prior to the priority date is confirmed;

- when using the proposed vaccines against PRRS and PVIS achieved the technical result provided for by the task of creating the invention.

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

Information sources

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Table 1
Immunogenic activity of emulsion inactivated vaccine against PRRS and PVIS Gilt Antibody level The results of the control infection with PRRS virus 35 days after vaccination before vaccination 34 days after vaccination against PRRS (in IFA, S / P) against PVIS (in RTGA) against PRRS (in IFA, S / P) against PVIS (in RTGA) one 0.04 1:64 0.55 1: 2048 - 2 0,03 1: 128 0.90 1: 2048 - 3 0,07 1: 128 0.51 1: 1024 - four 0.05 1:64 0.35 1: 2048 - 5 (control) 0.05 1:64 0.06 1:64 + 6 (control) 0.06 1:64 0,07 1:64 + Note: "+" - clinical signs of the disease;
"-" - lack of clinical signs of the disease;
S / P less than 0.3 - negative result;
S / P is 0.3-0.4 - a dubious result;
S / P greater than 0.4 - a positive result. table 2
Immunogenic activity of emulsion inactivated vaccine against PRRS and PVIS Gilt Antibody level The results of the control infection with PRRS virus 35 days after vaccination before vaccination 34 days after vaccination against PRRS (in IFA, S / P) against PVIS (in RTGA) against PRRS (in IFA, S / P) against PVIS (in RTGA) one 0.01 1:64 0.65 1: 1024 - 2 0.08 1:32 0.40 1: 2048 - 3 0.01 1:32 0.97 1: 1024 - four 0.02 1:64 0.40 1: 2048 - 5 (control) 0.02 1:64 0.06 1:64 + 6 (control) 0,07 1:64 0.02 1:64 + Note: "+" - clinical signs of the disease;
"-" - lack of clinical signs of the disease;
S / P less than 0.3 - negative result;
S / P is 0.3-0.4 - a dubious result;
S / P greater than 0.4 - a positive result.

Table 3
Immunogenic activity of emulsion inactivated vaccine against PRRS and PVIS Gilt Antibody level The results of the control infection with PRRS virus 35 days after vaccination before vaccination 34 days after vaccination against PRRS (in IFA, S / P) against PVIS (in RTGA) against PRRS (in IFA, S / P) against PVIS (in RTGA) one 0.08 1:32 0.40 1: 2048 - 2 0.04 1:32 0.30 1: 2048 - 3 0.05 1:32 0.97 1: 1024 - four 0.02 1:32 1.20 1: 2048 - 5 (control) 0,03 1:32 0.05 1:64 + 6 (control) 0.06 1:64 0.06 1:64 + Note: "+" - clinical signs of the disease;
"-" - lack of clinical signs of the disease. Table 4
Indicators of immunogenic activity of the associated vaccine against PRRS and PVIS in experiments on gilts and rabbits Vaccine sample no. Sod. Hypertension, (%) Animals Anti-virus antibody titers view qty PRRS (RNGA) PVIS (RTGA) before vacc. 30 days after vaccine. before vacc. 30 days after vaccine. one 43 rabbits gilts 5
3 <1:20 <1:20 448 ± 78 853 ± 213 115 ± 12
<1:32 512 ± 0
1024 ± 0 2 45 rabbits gilts 5
3 <1:20 <1:20 480 ± 101 426 ± 107 89 ± 15
<1:32 614 ± 102
1126 ± 198 3 47 rabbits gilts 5
3 <1:20 <1:20 512 ± 78 533 ± 106 51 ± 8
<1:32 1024 ± 0
1365 ± 341

Table 5
Indicators of immunogenic activity of emulsion inactivated vaccine against PRRS and PVIS on gilts Gilt Antibody level The results of the control infection with PRRS virus 35 days after vaccination before vaccination 35 days after vaccination against PRRS (in IFA) against PVIS (in RTGA) against PRRS (in IFA) against PVIS (in RTGA) one 0.04 1:64 0.55 1: 2048 - 2 0,03 1: 128 0.90 1: 2048 - 3 0,07 1: 128 0.51 1: 1024 - four 0.05 1:64 0.35 1: 2048 - 5 0.01 1: 128 0.48 1: 1024 - 6 0.01 1: 128 0.43 1: 1024 - 7 0,03 1: 128 0.34 1: 1024 - 8 0.05 1:64 0.67 1: 512 - 9 0.06 1:64 0.28 1: 1024 - M ± m 0.04 ± 0.01 99 ± 18 0.50 ± 0.06 1308 ± 193 the control 10 0.05 1:64 0.06 1:64 + eleven 0.06 1:64 0,07 1:64 + 12 0.01 1: 128 0.02 1: 128 + Note: The value of S / P in ELISA ≥0.4 - the result is positive;
"+" - the presence of a temperature reaction and typical clinical manifestations;
"-" - lack of temperature reaction and clinical manifestations. Table 6
Indicators of post-vaccination immunity in pigs of different sex and age groups vaccinated with inactivated emulsion vaccine against PRRS and PVIS Group of animals Number of animals Antibody level, after 55-70 days against viruses PRRS (IFA) PVIS (RTGA) Main sows twenty 217 ± 29 3022 ± 383 Repair pigs 16 294 ± 32 3968 ± 597 Boars 12 208 ± 43 3925 ± 640 Weaned piglets 24 299 ± 32 1653 ± 262 Note: The value in the ELISA: <1:50 - negative result;
> 1: 100 is a positive result.
Value in RTGA <1:64 - negative result;
> 1: 128 - a positive result.

Table 7
Antibody titers in sows immunized against PRRS and PVIS Households Age group of animals Number of samples The average level of antibodies against viruses PRRS (RNGA) PVIS (RTGA) sows: before insemination 19 544 ± 66 2157 ± 342 JSC "Silent Don" 2nd floor. gestation 26 586 ± 53 2389 ± 378 after farrowing 10 338 ± 74 1706 ± 264 pigs "domolosive" 10 <1:20 <1:32 sows: before insemination eighteen 391 ± 45 1678 ± 263 Pig farm 2nd floor. gestation after farrowing 17 753 ± 83 2078 ± 305 Kuznetsovsky eighteen 271 ± 50 1614 ± 290 pigs "domolosive" 10 <1:20 <1:32 Sows: before insemination eleven 524 ± 61 1914 ± 387 OJSC 2nd floor. gestation 13 837 ± 85 2934 ± 503 "Ilyinogorsky" after farrowing 8 500 ± 70 1184 ± 210 pigs "domolosive" 5 <1:20 <1:32 sows: before insemination fifteen 395 ± 54 2014 ± 394 CJSC "Nadeevo" 2nd floor. gestation 17 616 ± 79 2409 ± 328 after farrowing twenty 238 ± 69 1152 ± 205 pigs "domolosive" 9 20 ± 0 <1:32 sows: before insemination 6 350 ± 80 1707 ± 215 Pig farm 2nd floor. gestation after farrowing 9 675 ± 82 1820 ± 444 Vladimirsky 9 568 ± 47 1223 ± 399 pigs "domolosive" 5 24 ± 4 <1:32

Table 8
The effectiveness of the use of emulsion inactivated vaccines against PRRS and PVIS in households disadvantaged for these diseases Households Duration of vaccine use, (months) The level of reproductive pathology,% before vaccine after vaccine JSC "Vishnevskoe" 24 78.8 3.0 JSC "Silent Don" 24 74.0 11.8 Pig complex "Kuznetsovsky" 5 2 * CJSC "Nadeevo" 6 70 2 Pig complex "Lazarevsky" 24 14.1 * APP named after Kirov 12 79.3 * AOZT Kommunar 6 66.6 * Note: * - isolated cases.

Claims (14)

1. The vaccine associated emulsion inactivated against reproductive-respiratory syndrome and porvovirus infection of pigs, containing antigenic material from the causative agent of parvovirus infection of pigs, reproduced in a sensitive biological system and subjected to inactivation, and an oil adjuvant, characterized in that as an antigenic material from the pathogen infection pigs it contains antigenic material from the strain "Vl-94-DEP" of parvovirus pigs, a collection of VGNKI "Vl-94-DEP", and additionally antigenic material from the strain "BD-DEP" reproductive and respiratory syndrome of pigs, collection VGNKI "BD-DEP", in an effective amount. 2. The vaccine according to claim 1, characterized in that it contains antigenic material from the strain "BD-DEPT" of the virus of reproductive and respiratory syndrome of pigs reproduced in transplantable cell culture Marc-145. 3. The vaccine according to claim 2, characterized in that it contains antigenic material from the strain "BD-DEPT" of the virus of reproductive and respiratory syndrome of pigs, reproduced in transplantable cell culture Marc-145 with biological activity of at least 6.0 lg TCD ₅₀ / cm ³ . 4. The vaccine according to any one of claims 1 to 3, characterized in that it contains antigenic material from the strain "DB-DEPT" of the virus of reproductive and respiratory syndrome of pigs reproduced in a transplantable cell culture of Marc-145 with biological activity of at least 6.0 lg TCD ₅₀ / cm ³ and inactivated by aminoethylethyleneimine, in an amount of 30.0-32.0 wt.%. 5. The vaccine according to claim 1, characterized in that it contains antigenic material from the strain "Vl-94-DEP" of porcine parvovirus reproduced in a transplantable cell culture of PPC. 6. The vaccine according to claim 5, characterized in that it contains antigenic material from the strain "Vl-94-DEP" of porcine parvovirus, reproduced in an inoculated cell culture of PPC with biological activity of at least 7.5 lg TCD ₅₀ / cm ³ . 7. The vaccine according to claim 5 or 6, characterized in that it contains antigenic material from the Vl-94-DEP strain of porcine parvovirus reproduced in an inoculated cell culture of PPC with hemagglutinating activity of at least 1: 2048 GAE. 8. The vaccine according to claim 7, characterized in that it contains antigenic material from the strain "Vl-94-DEP" of porcine parvovirus, reproduced in an inoculated cell culture of PPC with biological activity of at least 7.5 lg TCD ₅₀ / cm ³ and GA activity of at least 1: 2048 GAE and inactivated by aminoethylethyleneimine, in an amount of 15.0-17.0 wt.%. 9. The vaccine according to claim 1, characterized in that it contains antigenic material from the strain "BD-DEPT" of the virus of reproductive and respiratory syndrome of pigs and antigenic material from the strain "VL-94-DEPT" of the pig parvovirus in a ratio of at least 2: 1. 10. The vaccine according to claim 1, characterized in that of the oil adjuvants it contains an oil adjuvant ARRIAH. 11. The vaccine of claim 10, characterized in that it contains an oil adjuvant ARRIAH in an amount of at least 53.0 wt.%. 12. The vaccine according to claim 1, characterized in that of the oil adjuvants it contains an oil adjuvant "Montanide IZA-70". 13. The vaccine according to item 12, wherein the oil adjuvant contains oil adjuvant "Montanide IZA-70" in an amount of at least 53%. 14. The vaccine according to claim 1, characterized in that it contains antigenic material from the strain "BD-DEPT" of the virus of reproductive and respiratory syndrome of pigs reproduced in transplantable cell culture Marc-145 with biological activity of at least 6.0 lg TCD ₅₀ / cm ³ and inactivated by aminoethylethyleneimine, antigenic material from the Vl-94-DEP strain of porcine parvovirus reproduced in a transplantable cell culture of PPC with biological activity of at least 7.5 lg TCD ₅₀ / cm ³ and hemagglutinating activity, at least 1: 2048 GAE and otherwise ivirovannogo aminoetiletileniminom, and oil in a ratio of adjuvant, wt.%: Antigenic material from the strain "BD-DEP" swine reproductive and respiratory syndrome virus 30.0-32.0 Antigenic material from the strain Vl-94-DEP of pig parvovirus 15.0-17.0 Oil adjuvant Up to 100.0