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

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

 

The invention relates to the field of veterinary Virology and biotechnology and can be used in the design and manufacture of tools for the specific prevention of reproductive-respiratory syndrome and parvovirus infection of pigs.

Reproductive and respiratory syndrome swine (PRRS) and parvovirus infection of pigs (PVIS) is a highly contagious disease of swine characterized by impaired reproduction in sows, abortion, birth of dead or weak piglets with a high mortality rate.

PRRS was registered in the late 80-ies of the last century, first in farms in the U.S. and Canada, and then in many other countries. By the mid 90-ies was covered by almost all of Europe. In Russia and some other CIS countries PRRS registered since 1993.

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

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

PRRS has immunosuppressive properties and this creates the preconditions for the emergence of secondary viral or bacterial infections. It was established that in the majority of Vinodolski farms, disadvantaged by PRRS, simultaneously circulating the causative agents of several diseases, including pathogen PVIS, causing violations of animal reproduction (2).

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

The relationship of parvovirus pigs (PVA) with impaired reproductive function sows in vivo was first established in 1967 in the UK, however, experimentally the disease was reproduced in 10 years.

Disease in sows flows, usually without visible symptoms. There is only disorder of the reproductive function. The virus crosses the placenta and infects the fruit in the 1st half of gestation, causing their mummification and death. Clinically the disease manifests with pogolotti, early in the 1st half of gestation, abortion and premature birth. Upon the occurrence of the disease in previously prosperous farms in the birth of live piglets per sow per year is reduced by 50-60%in inpatient disadvantaged farms - 10-20%.

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

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

The advantage of live vaccines because of the technology in the manufacture and simplicity in the application there is no doubt, however, the immune response in pigs when their introduction is not always stable and in some cases causes complications after vaccination.

In the world of industrial pig for the specific prevention of PRRS and PVIS widely used emulsion inactivated single prepared from different strains of pathogens, reproduced in sensitive biological systems (4-13).

The main disadvantages of these vaccines are that they have low antigenic and immunogenic activity and/or are unsafe, as is used in the production virulent strains, which always create problems when performing the requirements of the veterinary-sanitary safety.

In addition, these vaccines against PRRS and PVIS are plain products and contain only one of the two vaccine strains and their subsequent independent introduction provides a stronger stress on the body of the immunized animals and requires twice the amount of labour costs in vaccination.

This disadvantage is partially resolved by the creation associated vaccines containing individually or in combination of PRRS virus antigens and PVIS the discrepancies between the different strains.

Known vaccine associated inactivated against PVIS and leptospirosis, containing antigens of Leptospira sero-groups Pomona and Tarassovi, vaccinated material strain Parvovirusporaina And-82 hemagglutinin activity (HA activity) 1:256-1:1512 AI/of 0.025 cm3selected in optimal proportions, as inactivant formalin or formalin and dimer etilenimina and adjuvant aluminium hydroxide and protein serum (14).

Known vaccine associated inactivated against PVIS and Aujeszky's disease, containing antigenic materials homologous pathogens in optimal ratios (15).

Known vaccine associated emulsion inactivated against circoviruses infection of pigs (CVIS) and PVIS containing antigenic material from strains of homologous viruses, reproduced in sensitive biological systems, inactivant and oil adjuvant in effective relation (16).

Known vaccine associated inactivated against PRRS, PVIS, leptospirosis and erysipelas containing antigenic materials homologous causative agents of these infections in optimal proportions (17).

The main disadvantages associated vaccines are in their low antigenic and immunogenic.

Closest to the proposed of which briteney on essential features is the vaccine emulsion inactivated against PVIS, containing antigenic material from strain And 82 homologous virus reproduced in sensitive biological system and subjected to inactivation, and oil adjuvant in effective relation (18).

The vaccine prototype has significant drawbacks:

- it has a low antigenic and immunogenic activity and does not provide satisfactory protection against the OIE PVS circulating on the territory of the Russian Federation;

- it does not create protection against PRRS;

as inactivate it contains formalin, causing destructive changes of viral proteins and reducing immunogenic activity of the vaccine.

In this regard, the problem of creating a vaccine associated emulsion inactivated against PRRS and PVIS, highly antigenic and immunogenic activity and emissions, continues to be relevant and is the main focus of research on how to improve them. The need for the development and production of these vaccines due to the fact that nine out of ten pig farms of the Russian Federation, troubled by PRRS, at the same time circulates the parvovirus. This is confirmed by the results of both domestic and foreign scientists.

In the task of creating the present invention was to develop Vysokomol the gene and harmless vaccine associated emulsion inactivated against PRRS and PVIS on the basis of new production strains homologous virus, allocated on the territory of the Russian Federation, which has high biological, antigenic and immunogenic activity in the native form and after inactivation and suitable for the manufacture of the associated emulsion inactivated drug creates intense and long-lasting immunity in vaccinated animals against circulating in Russia pathogens PRRS and PVIS.

The technical result from use of the present invention is to expand the Arsenal of vaccines associated emulsion inactivated against PRRS and PVIS with high immunogenic activity and emissions and creating intense and long-lasting immunity in vaccinated animals against circulating in Russia pathogens PRRS and PVIS.

This technical result is achieved by the creation of a vaccine associated emulsion inactivated against PRRS and PVIS, characterized by the following combination of characteristics:

1. Vaccine associated emulsion inactivated against PRRS and PVIS.

2. Antigenic material of the causative agent of PRRS, reproduced in sensitive biological system, in an effective amount.

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

2.2. Antigenic material is ial group of strain BD-DEPT" of PRRS virus, reproduced in transplantable cell culture Marc-145.

2.3. Antigenic material from strain "DB-DEPT" of PRRS virus reproduced in transplantable cell culture mags-145 with biological activity, at least 6,0 lg TCD50/cm3.

3. Inactivant.

3.1. Of inactivated aminoethylethanolamine (AAAI).

3.2. AEEI in a 1% aqueous solution.

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

4. Antigenic material from strain "DB-DEPT" of PRRS virus reproduced in transplantable cell culture Marc-145 with biological activity, at least 6,0 lg TCD50/cm3and inactivated AAAI, the number 30,0-32,0 wt.%.

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

5.1. As antigenic material from the pathogen PVIS antigenic material from strain "VL-94-DEPT" PVS.

5.2. Antigenic material from strain "VL-94-DEPT" PVA reproduced in transplantable cell cultures kidney pig ACC.

5.3. Antigenic material from strain "VL-94-DEPT" PVA reproduced in transplantable cell cultures kidney pig CPD with a biological activity of at least 7,5 lg TCD50/cm3.

5.4. Antigenic material from strain "VL-94-DEPT" PVA reproduced in transplantable cell culture CPD with HA activity, p is at least 1:2048 GUY.

6. Inactivant.

6.1. Of inactivated AAAI.

6.2. AEEI in a 1% aqueous solution.

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

7. Antigenic material from strain "VL-94-DEPT" PVA reproduced in transplantable cell culture CPD with a biological activity of at least 7,5 lg TCD50/cm3and HA activity of at least 1:2048 GUY and inactivated AAAI, in the number - 15,0-17,0 wt.%.

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

9. Oil adjuvant.

9.1. From the oil adjuvants oil adjuvant ARRIAH (19).

9.2. Oil adjuvant ARRIAH in number at least to 53.0 wt.%.

9.3. From the oil adjuvants oil adjuvant "Montanide ISA-70".

9.4. Oil adjuvant "Montanide ISA-70 in number, at least, to 53.0 wt.%.

10. A mixture of antigenic materials from strain "DB-DEPT" of PRRS virus reproduced in transplantable cell culture Marc-145 with biological activity, at least 6,0 lg TCD50/cm3and inactivated AAAI, and strain VL-94-DEPT" PVA reproduced in transplantable cell culture CPD with a biological activity of at least 7,5 lg TCD50/cm3and HA activity of at least 1:2048 GUY and inactivated AAAI, combined with an oil adjuvant rela is Oseni, at least 47:53.

11. Antigenic material from strain "DB-DEPT" of PRRS virus reproduced in transplantable cell culture Marc-145 with biological activity, at least 6,0 lg TCD50/cm3and inactivated AAAI, antigenic material from strain "VL-94-DEPT" PVA reproduced in transplantable cell culture CPD with a biological activity of at least 7,5 lg TCD50/cm3and HA activity of at least 1:2048 GUY and inactivated AAAI, and oil adjuvant at the ratio, wt.%:

Antigenic material from strain
"DB-DEPT" of PRRS virus30,0-32,0
Antigenic material from strain
"VL-94-DEPT" PVS15,0-17,0
Oil adjuvantTo 100.0

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

1. Vaccine associated emulsion inactivated against PRRS and PVIS.

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

3. Antigenic material from strain "VL-94-DEPT" PVIS reproduced in sensitive biological system and subjected to inactivation, in an effective amount.

4. Oil adjuvant.

The invention is also characterized by other symptoms, expressing a particular form of execution or specific conditions of its use:

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

2. Antigenic material from strain "DB-DEPT" of PRRS virus reproduced in transplantable cell culture Marc-145 with biological activity, at least 6,0 lg TCD50/cm3.

3. Antigenic material from strain "DB-DEPT" of PRRS virus reproduced in transplantable cell culture mags-145 with biological activity, at least 6,0 lg TCD50/cm3and inactivated AAAI, the number 30,0-32,0 wt.%.

4. Antigenic material from strain "VL-94-DEPT" PVA reproduced in transplantable cell culture CPD.

5. Antigenic material from strain "VL-94-DEPT" PVA reproduced in transplantable cell culture CPD with a biological activity of at least 7,5 lg TCD50/cm3.

6. Antigenic material from strain "VL-94-DEPT" PVA reproduced in transplantable cell culture CPD with HA activity of at least 1:2048 GUY

7. Antigenic material from strain "VL-94-DEPT" PVA reproduced in transplantable cell culture CPD with a biological activity of at least 7,5 lg TCD50/cm3and HA activity of at least 1:2048 GUY and inactivated AAAI, the number 15,0-17,0 wt.%.

8. Antigenic material from strain "DB-DEPT" of PRRS virus and antigenic material from strain "VL-94-DEPT" PVA ratio of at least 2:1.

9. From the oil adjuvants oil adjuvant ARRIAH.

10. Oil adjuvant ARRIAH in number at least to 53.0 wt.%.

11. From the oil adjuvants oil adjuvant "Montanide ISA-70".

12. Oil adjuvant "Montanide ISA-70 in number, at least, to 53.0 wt.%.

13. Antigenic material from strain "DB-DEPT" of PRRS virus reproduced in transplantable cell culture Marc-145 with biological activity, at least 6,0 lg TCD50/cm3and inactivated AAAI, antigenic material from strain "VL-94-DEPT" PVA reproduced in transplantable cell culture CPD with a biological activity of at least 7,5 lg TCD50/cm3and HA activity of at least 1:2048 GUY and inactivated AAAI, and oil adjuvant at the ratio, wt.%:

Antigenic material
from strain "DB-DEPT virus RR is 30,0-32,0
Antigenic material
from strain "VL-94-DEPT" PVS15,0-17,0
Oil adjuvantTo 100.0

Features of the invention, characterizing the proposed vaccine that matches the characteristics of the prototype, including a generic term that reflects the assignment are:

1. The emulsion vaccine inactivated against PVIS.

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

3. Oil adjuvant.

Compared with vaccine-the prototype of the significant distinguishing features of the proposed vaccine are that as antigenic material from the pathogen PVIS it contains antigenic material from strain "VL-94-DEPT" PVS and additionally antigenic material from strain "DB-DEPT" of PRRS virus in an effective amount.

The invention is also characterized other distinctive signs, expressing a particular form of execution or specific conditions of its use:

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

2. Antigenic material from strain "DB-DEPT" of PRRS virus reproduced in transplantable cell culture Marc-C biological activity, at least 6,0 lg TCD50/cm3.

3. Antigenic material from strain "DB-DEPT" of PRRS virus reproduced in transplantable cell culture Marc-145 with biological activity, at least 6,0 lg TCD50/cm3and inactivated AAAI, the number 30,0-32,0 wt.%.

4. Antigenic material from strain "VL-94-DEPT" PVA reproduced in transplantable cell culture CPD.

5. Antigenic material from strain "VL-94-DEPT" PVA reproduced in transplantable cell culture CPD with a biological activity of at least 7,5 lg TCD50/cm3.

6. Antigenic material from strain "VL-94-DEPT" PVA reproduced in transplantable cell culture CPD with HA activity of at least 1:2048 GUY.

7. Antigenic material from strain "VL-94-DEPT" PVA reproduced in transplantable cell culture CPD with a biological activity of at least 7,5 lg TCD50/cm3and HA activity of at least 1:2048 GUY and inactivated AAAI, the number 15,0-17,0 wt.%.

8. Antigenic material from strain "DB-DEPT" of PRRS virus and antigenic material from strain "VL-94-DEPT" PVA ratio of at least 2:1.

9. From the oil adjuvants oil adjuvant ARRIAH.

10. Oil adjuvant ARRIAH in number at least to 53.0 wt.%.

11. From the oil adjuvants oil adjuvant "Montanide ISA-70".

p> 12. Oil adjuvant "Montanide ISA-70 in number, at least, to 53.0 wt.%.

13. Antigenic material from strain "DB-DEPT" of PRRS virus reproduced in transplantable cell culture Marc-145 with biological activity, at least 6,0 lg TCD50/cm3and inactivated AAAI, antigenic material from strain "VL-94-DEPT" PVA reproduced in transplantable cell culture CPD with a biological activity of at least 7,5 lg TCD50/cm3and HA activity of at least 1:2048 GUY and inactivated AAAI, and oil adjuvant at the ratio, wt.%:

Antigenic material
from strain "DB-DEPT" of PRRS virus30,0-32,0
Antigenic material
from strain "VL-94-DEPT" PVS15,0-17,0
Oil adjuvantTo 100.0

The proposed vaccine expands the Arsenal of tools for the specific prevention of PRRS and PVIS.

The achievement of the technical result from use of the present invention is explained by the fact that the associated emulsion inactivated vaccine against PRRS and PVIS entered antigenic materials from new strains of pathogens infections: antigenic material from strain "DB-DEPT" the RCC and antigenic material from strain "VL-94-DEPT" PVS, - with high biological, antigenic and immunogenic activity in the native form and after inaktivirovanie and providing vaccines that create intense and long-lasting immunity in vaccinated animals.

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

Additional technical result from the use of the invention is achieved by the fact that inactivation vaccinated material used AAAI that can significantly reduce labor and energy costs in the manufacture of vaccines and to improve the quality of antigenic material.

Additional technical result from use of the present invention to improve the antigenic and immunogenic activity of the target product can also be achieved by using in its composition an oil adjuvant ARRIAH or Montanide ISA-70".

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

The original virus to obtain strain "BD" separated from the blood serum of pigs with clinical signs of PRRS obtained from the former collective farm "Rassvet" Kirovsky district of the Stavropol territory in 1996. Production strain "DB" obtained by multiple serial passages in 3-day cultures the cells Marc-145.

Strain "DB" deposited in the all-Russian state collection of strains of microorganisms used in veterinary medicine and animal husbandry, all-Russian state research Institute for control, standardisation and certification of veterinary preparations (VGNKI) 25 may 1998 under registration code "DB-DEPT".

Strain "DB-DEPT" is harmless and has a high biological, antigenic and immunogenic activity in the native form and after inactivation. Experimentally confirmed the possibility of its use for the manufacture of vaccines.

Strain "DB-DEPT virus RRSSS characterized by the following features and properties.

Morphological properties

Strain "DB-DEPT" of PRRS virus belongs to the family Arteriviridae, genus Arterivirus, RNA-genome, has morphological features typical of PRRS: the shape of the virion is spherical, the size of the virion 45-75 nm with a core filling 3/4 of the virion diameter of 25-35 nm, surrounded by a lipid shell.

Antigenic properties

The PRRS virus strain "DB-DEPT" stable neutralized homologous anticorodal. The virus has no hemagglutination properties. When the vaccine virus induces the formation of specific antibodies detected in the enzyme-linked immunosorbent assay (ELISA). Polymerase chain reaction (PCR) revealed the city of the Autonomous RNA virus PRRS. Nucleotide sequencing of strain BD-DEPT" of PRRS virus showed significant (about 40%) in contrast to the strain CNCM No. 1-1102 and strain ATS VR-2332, a recognized reference in Europe and North America respectively. Identification and specificity of PRRS virus was determined in ELISA (IDEXX, USA) with a control of specificity. The result of differential diagnosis was negative for the virus Aujeszky's disease, swine parvovirus, a virus diarrhea of cattle and the virus transmissible gastroenteritis.

Biotechnological characteristics

Strain "DB-DEPT" shows high biological, antigenic and immunogenic activity. Strain "DB-DEPT" is intended for the preparation of diagnostic and vaccine preparations. Strain "DB-DEPT" of PRRS virus reproducerea in the cytoplasm of cells of 3-day cell culture Marc-145 at a temperature of 37°C. the Original suspension of the serum containing the virus infected cell culture Marc-145. After conducting 7-10 passages within 48-72 hours of incubation the virus accumulates to a titer of 4.0 to 4.5 lg TCD50/cm3.

Virus infectivity was enhanced by serial rapidly alternating passages, which passage 41 has reached 7,0-7,5 lg TCD50/cm3. Strain "DB-DEPT is stable and harmless.

Chemo - and geotectonically feature

Strain "DB-DEPT" is an RNA-containing virus. kleinova acid contains one thread, the surface of the virus covered by a layer of glycoproteins. The protein shell detects the following fragments:

M - transmembrane protein;

E - membrane protein;

GL- surface glycoprotein;

GS- surface glycoprotein;

N - nucleocapsid protein.

Physical properties

The mass of the virion from 49×103Yes up to 55×103Yes. Buoyant density gradient of cesium chloride 1,18-1.2 g/cm3.

Resistance to external factors

Strain "DB-DEPT" not resistant to solvents (ether, chloroform) and detergents that are sensitive to formaldehyde, ultraviolet irradiation, gamma irradiation and desiccation.

Additional characteristics and properties

Immunogenic activity within 14 days after immunization of pigs causes the formation of specific antibodies.

Reactogenicity is missing.

Pathogenicity is missing.

Virulence is missing.

Carcinogenesis is missing.

Contagiousness is missing.

The stability of attenuation installed when conducting 5 passages on the sensitive pigs.

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

The strain VL-94 is the new, previously unknown. The original virus to obtain strain VL-94" highlighted in the research Institute of animal protection in 1994 when virological examination of the internal organs of an aborted (up to 15 cm long) fruit sows brought from the farm "Vladimir, Vladimir region. The production strain VL-94" PVA obtained by serial passages on transplantable cell cultures kidney pig "Kazan" (CPD).

The resulting strain deposited at the Russian state collection of strains of microorganisms used in veterinary medicine and animal husbandry, all-Russian state research Institute for control, standardisation and certification of veterinary preparations (VGNKI) of the Ministry of agriculture of the Russian Federation on 1 July 1996 under registration code "VL-94-DEPT".

The strain VL-94-DEPT" PVA has a high biological, antigenic and immunogenic activity in the native form and after inactivation and has high productivity in sensitive biological systems of cultivation. Experimentally confirmed the possibility of its use for the preparation of vaccines, creating intense and long-lasting immunity in vaccinated animals.

The strain VL-94-DEPT" PVS is characterized by the following characteristics and properties.

Morphological properties

The strain VL-94-DEPT" PVS is tositsa to the family Parvoviridae, the genus Parvovirus. Mature virion has a cubic symmetry, includes 32 capsomere, 2 or 3 capsid protein, the diameter of the virion is about 20 nm, does not contain membranes and lipids, mol. weight is 5.3×106Yes. The viral genome presents 1-stranded DNA mol. weight of 1.4 kDa, i.e. 26.5% of the mass of the complete virion, the proportion of guanine + cytosine is 41÷53%. Buoyant density in CsCl full of infectious virus, incomplete ("empty") of the virion and extracted mirinoi DNA at 1.38÷1,395; 1,30÷1,315 and 1,724 g/cm3respectively. The sedimentation coefficient 110÷122 S.

Antigenic properties

The virus contains three polypeptide a, b and C (mol. weight 60÷90 kDa. All sravnivayete isolates PVA pigs were antigenically similar, if not identical. Their identity is established in PH, rtha. The virus has expressed antigenic activity and induces the synthesis of neutralizing, complimentative and precipitiously antibodies, as well as antihemagglutinin. The study of the antigenic properties of isolated individual virionyx polypeptides of the pathogen showed that anticavity to each structural protein will neutralize infectious activity of complete virions and interacts with all three structural proteins of the virus. Each individual protein interacts with virusneutralizing the sponding antibodies, obtained on the full introduction of the virus. Virousspecificakih antigenic determinants are presumably all virionyx proteins PVA. The biological half-life of antibodies in piglets depends on their mass, the average is 18 to 20 days.

Biotechnological characteristics

The strain VL-94-DEPT" PVS well reproduced in the transplantable cell culture CPD and accumulates in the title of 8.0 lg TCD50/cm3. Transplantable cell culture spew insensitive to the virus. A feature of the cultivation and accumulation of the virus is its dependence on cell division. Curve propagation of the virus depends on the number of mitoses. He is well reproduced in the dividing cells of the kidney of the fetal pig when infection within 48 hours after planting culture. The highest concentration of the virus is achieved in 2-3 days of cultivation. JRS is characterized by vacuolation and rounding of cells, diffuse granulation. When studying the breeding cycle method immunofluorescence revealed that the antigen is found in the nucleus of the cells after 6 hours after infection and stored in maximum quantities for 18-24 hours. The strain VL-94-DEPT" PVS is intended for manufacture of inactivated vaccines against PVIS. The strain VL-94-DEPT" PVS is genetically stable and retains immunobiological properties ove the position 10 passages (observation period).

Chemo - and geotectonically feature

Genome PVA strain "VL-94-DEPT" presents 1-spiral linear DNA molecule with molm 1,4 kDa. 1-spiral DNA consists of about 5 thousand nucleotides and encodes 4 protein - 3 structural and 1 non-structural. On both ends of the DNA are not identical, semicomplete sequence of nucleotides (palindromes), forming 2-helical hairpins that are resistant to nuclease S1; 3'-terminal hairpin consists of 115÷116 nucleotides, the 5'-end - from 200÷242 nucleotides. Hairpin structures play an important role in DNA replication. The genes encoding the structural proteins are located in the 5'-terminal half, and the gene encoding non-structural protein 3'-terminal half of the DNA. In the virions of strain VL-94-DEPT" contains negative DNA. The virus strain VL-94-DEPT" is a stand-alone (non-defective).

Pathogenic properties

The strain VL-94-DEPT" PVA pathogenic for cattle and apathogenic for hamsters and mice. All infected piglets symptoms and pathological changes are usually absent, however, are detected specific antibodies. The virus isolated from the seminal vesicles on day 7 to day 9 after infection. With 12 days in the serum of animals reveal antihemagglutinin. In piglets, were in contact with infected animals are detected specific antibodies that have Alstom about the contagiousness of the infection. After intracerebral, oral and intranasal infection in piglets specific reproducible clinical symptoms were observed. However, these animals were able to isolate the virus from stool, blood and most tissues. To 7 days were allocated specific antibodies. When infection of pigs with the virus of strain VL-94-DEPT" at different stages of pregnancy were observed transplacental infection of fetuses.

Hemagglutinins properties

The strain VL-94-DEPT" PVA agglutinate erythrocytes of Guinea-pig, chicken, cat, rat, mouse, monkey, human and not agglutinate erythrocytes of sheep, horses, pigs, cattle, dog, rabbit, hamster and ducks. To determine the HA activity using Guinea pig erythrocytes, the reaction is carried out at 4°and as the diluent used saline solution, pH of 7.2. HA activity in DSA was 1:2048 GUY. The virus is not released from erythrocytes at 37-40°C for one hour, but in alkaline buffer (pH 9,0) he fully aluinum with them for 30 minutes at room temperature.

Physical properties

The molecular mass of the virion is 5.5 to 6.2 Hmm, buoyant density in CsCl gradient 1,39÷1.42 g/cm3the sedimentation coefficient 110÷122 S.

Resistance to external factors

The strain VL-94-DEPT" PVA resistant to the action of various physico-helices what their factors. Retains infectivity at pH 3÷9 and 37°C for 1.5 hours, but completely inactivated at pH 2 under the same conditions. The virus is stable at 56°C for 2 days at 70° - 2 hours and at 80°With inactivated for 5 minutes without losing infectivity in culture fluid with 20% fetal cattle serum at 35÷37°With over 15 weeks. Sustainable handling chloroform, ether and freon in 10-50% concentration for 10÷30 min at room temperature. The virus is resistant to the action of RNA - and DNA-ases and proteases (papain, chemotrypsin, trypsin, pepsin), resistant to the action of ultrasound, is inactivated by UV rays, formalin, ethylenimines and its derivatives: β-propiolactone, glutaraldehyde. The virus remains active for more than 4 months, in pens of up to 4÷6 months.

Additional characteristics and properties

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

On the basis of the obtained data it can be argued that the strain VL-94-DEPT" antigenic and immunological spectra is original, in taxonomic relation to new, previously unknown isolate PVS.

Conducted by the applicant's analysis of the prior art, including searching by the patent and scientific and technical information sources and identify sources that contain information about the analogues PR is degenova of the invention, has allowed to establish that the applicant has not found the source, characterized by signs, identical to all the essential features of the present invention. The definition from the list of identified unique prototype as the most similar set of features analogue has allowed to establish the essential towards perceived by the applicant to the technical result of the distinctive features of the proposed vaccine set forth in the independent claim.

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

To check whether a proposed solution to the condition of patentability "inventive step" conducted an additional search of the known solutions to identify topics included in the characterizing portion of the independent claim. The search results showed that the proposed solution does not follow for the expert in the obvious way from the prior art, as set out in the description section (not identified solutions that have the signs consistent with the distinctive features of the present invention), and revealed no effect provided the essential features of the invention transformations to achieve a technical result.

The trail is 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 strain "DB-DEPT" of PRRS virus for the manufacture of the proposed vaccine is prepared from Matrosovo virus grown in human cell cultures Marc-145 3-days-old with a cell concentration of more than 100 tuscl/cm3. Matrawy virus is considered to be suitable for producing viral raw material if it meets the following requirements: the titer of infectivity after reproduction in the monolayer of cells Marc-145 6,5 lg TCD50/cm3pH of 7.2-7.4 and in the absence of contamination. For the manufacture of 20 liters of cultural production PRRS take 110-120 mattresses cell culture capacity of 1.5 DM3each. After draining the growth environment and a single laundering FBI or nutrient medium in mattresses make a 5 cm3vaccinated material motroway series. The adsorption of the virus was performed at 37°within hours. Then in mattresses make the environment a Needle in a volume of 150-200 cm3with the addition of 5% fetal cattle serum with antibiotics (gentamicin at a concentration of 50 µg/cm3or its analogues).

The cultivation is carried out at 37°With in 48-72 hours. For contrainsurgencia leave 3 of the mattress, which replaces the environment. The replication of the virus in cell culture Marc-145 determine the nature of the JRC with the formation of clusters of spherical cells, rising above the monolayer. In the control mattresses should not be any destructive changes of the cells. Mattresses that are observed cytopathic changes from the defeat to 60% of the cell monolayer (usually 48-72 hours), selected and frozen at -20°C. the Virus for the manufacture of vaccines get 2x freezing and thawing of infected cells with subsequent discharge of the contents of mattresses in one tank, observing sterile conditions. The resulting virus release from the cell debris by centrifugation at 1000 rpm./min for 10 minutes. Purified from cell detritus viral suspension should be transparent liquid pinkish-red color. Then from the tank take a sample for laboratory and production control infectious activity and sterility.

Production series of PRRS virus is considered suitable if it meets the following requirements: the titer of infectivity after reproduction in the monolayer of cells Marc-145 at least 6,0 lg TCD50/cm3pH of 7.2-7.4 and in the absence of contamination.

Purified vaccinated suspension is subjected to inactivation. Inactivation gained viral raw materials of the Vedas is t using a 1% aqueous solution AAAI, made up to a final concentration of 0.001 to 0.05%. For this purpose, the suspension is heated up to 26-28°To contribute with constant stirring solution AAAI and set the pH value of 7.6 to 7.8 by adding a 5% solution of succinic acid. Inactivation lead to thermostat at the temperature of (37±2)°C for 20-22 hours with periodic mixing. After inactivation, the suspension is cooled to 4-6°set the value of its pH in the range of 7.6-7.8 and take samples of the antigen to test for sterility, the avirulence and immunogenic activity, using the well-known specialist methods.

Example 2.

Antigenic material from strain "VL-94-DEPT" PVS for manufacturing the offered vaccine is prepared from Matrosovo virus adapted to transplantable cell cultures of kidney pig CPD grown on a nutrient medium parietal (SRP), and manifesting infectious activity in cell culture of CPD, at least 7,5 lg TCD50/cm3and HA activity of at least 1:2048 GUY. To obtain the antigenic material used grown in mattresses cell culture CPD with a good monolayer 2-day age. To obtain 20 liters of suspension PVA take 110-120 mattresses cell culture capacity of 1.5 DM3each. After draining the growth environment in mattresses contribute 5-7 cm3PVA motroway of the seed. After 40-6 minutes incubation at a temperature of (37± 2)°C mattresses make 150-200 cm3supporting medium (without serum of cattle). Your mattress is placed in a thermostat for culturing at a temperature of (37±2)°C. the Period of cultivation from 48 to 72 hours. To control uninfected leave 3 of the mattress, in which the growth medium is changed to 200 cm3supporting medium without serum. After 24 hours incubation the infected and control mattresses daily mikroskopiruyut. Mattresses that are observed cytopathic changes from the defeat to 60% of the cell monolayer (usually 48-72 hours), taken three times freeze at a temperature of not higher than -20°and thawed. Then vaccinated material cleared from cellular debris. To do this, the contents of mattresses poured into one container and chilled vaccinated suspension, observing sterile conditions, free from cellular debris by centrifugation at 1000 rpm for 10 minutes. Purified from cell detritus suspension should be transparent liquid pinkish-red color. The purified suspension contribute gentamicin (100 U) and stirred for 20 minutes Then out of the container take a sample for laboratory and production control for sterility, infection and HA activity of the virus. Production series PVS must be contagious, at least 7,5 lg TCD 50/cm3and HA activity of at least 1:2048 GUY. Purified vaccinated suspension is subjected to inactivation. Inactivation gained viral production lead using 1% aqueous solution AAAI. For this purpose, the suspension is heated up to 26-28°To contribute with constant stirring solution AEEI to a final concentration of 0.05-0.08% and set the pH value of 7.6 to 7.8 by adding a suspension of 5% solution of succinic acid. Inactivation lead to thermostat at the temperature of (37±2)°C for 20-22 hours with periodic mixing. After inactivation, the suspension is cooled to 4-6°To set the pH value of 7.6-7.8 and take samples of antigenic material to test for sterility, the avirulence and HA activity using well-known specialist methods.

Example 3.

Vaccine associated emulsion inactivated against PRRS and PVIS is prepared by dispersing a mixture of antigenic materials from strain "DB-DEPT" of PRRS virus and strain VL-94-DEPT" 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, ie 2 parts of antigenic material of PRRS virus and 1 part of PVA, which corresponds to the content in the proposed vaccine, wt.%:

Antigenic material
from strain "DB-DEPT" of PRRS virus30,0-32,0
Antigenic material
from strain "VL-94-DEPT" PVS15,0-17,0

Then the resulting mixture emuleret with oil adjuvant in a ratio of 47:53. As oil adjuvant use oil adjuvant ARRIAH or oil adjuvant "Montanide ISA-70" (company Seppic, France), as immunostimulating activity they are identical.

The finished vaccine is collected in a sterile container, Packed in sterile glass vials and control in accordance with the technical specifications.

The vaccine is an emulsion of white or white with a pink tint, slightly viscous consistency. During long-term storage (over 12 months), a slight detachment of mineral oil over nerastraivaisya homogeneous emulsion. With shaking, the vaccine becomes homogeneous structure.

Received the vaccine has an optimal component composition, wt.%:

Antigenic material from strain "DB-DEPT"
the PRRS virus, reproduced in transplantable
cell culture Marc-145 biological active the stew,
at least 6,0 lg TCD50/cm3
and inactivated AAAI30,0-32,0
Antigenic material from strain "VL-94-DEPT" PVS,
reproduced in transplantable cell culture
CPD with biological activity, at least,
7,5 lg TCD50/cm3and HA activity, at least,
1:2048 GUY and inactivated AAAI15,0-17,0
Oil adjuvantTo 100.0

The content of antigenic material in the vaccine in the above range is effective quantity in the product, ensuring the achievement of the technical result from the use of the invention.

The proposed vaccine is used for prophylactic purpose in disadvantaged and threatened by PRRS and PVIS farms. Sows, gilts and boars-producers vaccinated twice with an interval of 20-30 days for three weeks before mating (insemination). In subsequent previously immunized sows are vaccinated once for three weeks before mating (insemination), and boars manufacturers revaccinated every 6 months.

Piglets born from vaccinia is the R against PRRS and PVIS sows subjected to immunization 1.5-2 months of age, twice with an interval of 20-30 days. The vaccine is injected intramuscularly behind the ear in a dose of 2 cm3regardless of the age of animals.

Immunity in vaccinated animals comes at 21-30 days after vaccination and lasts for at least 6 months. The vaccine induces high levels of antibodies against PRRS and PVIS, provides reliable passive maternal immunity in young animals up to 20-25 days of age that allows you to protect him from the field virulent pathogens PRRS and PVIS in the early period of life.

Example 4.

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

Antigenic material from strain "DB-DEPT"
the PRRS virus, reproduced in transplantable
cell culture Marc-145 biological activity
6,0 lg TCD50/cm3and inactivated AAAI32,0
Antigenic material from strain "VL-94-DEPT" PVS,
reproduced in transplantable cell culture
CPD biological activity of 7.5 lg TCD50/cm3
and HA-activity 1:2048 AI
and inactivated AAAI15,0
Oil adjuvantTo 100.0

Immunogenic activity of the vaccine was determined in pigs with a live weight 30-40 kg 4-6 weeks of age, free from antibodies to PRRS virus and PVIS. The vaccine was administered at 2.0 cm3intramuscularly in the neck. In animals prior to vaccination and after 34 days after vaccination were selected blood samples and tested in ELISA (IDEXX kits, USA) for the presence of specific antibodies. Their evaluation was carried out according to the value of S/P against the PRRS virus and the titer of HA activity against PVS.

The research results are summarized in table 1.

From table 1 it is evident that in the blood serum of vaccinated animals appeared specific antibodies in high titers. Vaccinated animals do not get sick after controlling infecting their virulent reference PRRS virus, strain Lelystad".

Example 5.

Tested immunogenic vaccine associated emulsion inactivated against PRRS and PVIS based on the strains of "DB-DEPT" and "VL-94-DEPT", respectively, produced as described in examples 1, 2 and 3, and containing, m is S.%:

Antigenic material from strain "DB-DEPT"
the PRRS virus, reproduced
in transplantable cell culture Marc-145
biological activity of 6.5 lg TCD50/cm3
and inactivated AAAI30,0
Antigenic material from strain "VL-94-DEPT" PVS,
reproduced in transplantable cell cultures ACC
biological activity of 8.0 lg TCD50/cm3
and HA-activity 1:2048 GUY and inactivated AAAIof 17.0
Oil adjuvantTo 100.0

Immunogenic activity of the vaccine was determined in pigs with a live weight 30-40 kg 4-6 weeks of age, free from antibodies to PRRS virus and PVIS. The vaccine was administered at 2.0 cm3intramuscularly in the neck. In animals prior to vaccination and after 34 days after vaccination were selected blood samples and tested in ELISA (IDEXX kits, USA) for the presence of specific antibodies. Their evaluation was carried out according to the value of S/P against the PRRS virus and the titer of HA activity against PVS.

The results of the research the work presented in table 2.

The data in table 2 indicate that in the blood sera of animals vaccinated with a vaccine against PRRS and PVIS, revealed a high level of post-vaccination antibody.

Example 6.

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

Antigenic material from strain "DB-DEPT"
the PRRS virus, reproduced in transplantable
cell culture Marc-145 biological activity
7,0 lg TCD50/cm3and inactivated AAAI30,0
Antigenic material from strain
"VL-94-DEPT virus PVIS reproduced
in transplantable cell cultures ACC
biological activity of 7.5 lg TCD50/cm3
and HA-activity 1:2048 AI and
inactivated AAAIof 17.0
Oil adjuvantTo 100.0

managenow activity of the vaccine was determined in pigs with a live weight 30-40 kg 4-6 weeks of age, free from antibodies to PRRS virus and PVIS. The vaccine was administered at 2.0 cm3intramuscularly in the neck. In animals prior to vaccination and after 34 days after vaccination were selected blood samples and tested in ELISA (IDEXX kits, USA) for the presence of specific antibodies. Their evaluation was carried out according to the value of S/P against the PRRS virus and the titer of HA activity against PVS.

The results are shown in table 3.

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

Example 7.

For testing immunogenic offer vaccines produced three prototype drug with different concentrations of antigen and an oil adjuvant ARRIAH.

After checking for sterility and safety in mice each sample was injected intramuscularly in the thigh 5 rabbits on 1 cm3and three pigs in the neck 2 cm3. The animals were constant surveillance. The blood of animals was collected before and 30 days after vaccination. Monitoring of vaccinated animals showed that the vaccine is harmless, because any deviations in their condition was not noted. Serum antibodies against PRRS virus was investigated in RNA and against PVS - in rtga. The results of the research sivaram is the blood of the vaccinated pigs are shown in table 4.

According to table 4, all animals used in the experiment were seronegative, except for some rabbits, which in the serum were detected antibodies against PVA in low titers (≤1:128). After 30 days in the blood serum of all vaccinated animals showed specific antibodies against PRRS virus and PVIS. So, the pigs antibodies against PRRS virus in average in the groups ranged from 1:426±107 to 1:853±213, and in rabbits is from 1:448±78 to 1:512±78. Antibody titers against PVS were high and the average for the group was 1:1024±0-1:1365±341 of gilts and 1:512±0-1:1024±0 in rabbits. Antigen content from 43% to 47% did not significantly affect the immunogenicity of the vaccine.

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

Example 8.

The proposed vaccine was tested for immunogenic activity through the control of infection of immunized animals virulent PRRS virus, strain Lelystad. In addition, selected paired serum prior to vaccination and the PE the units control infection were investigated using a commercial ELISA test (IDEXX). The aggregated results of these experiments are presented in table 5.

According to table 5, six of the nine vaccinated pigs had over 35 days high level of antibodies in the blood against the PRRS virus. So, in ELISA ratio S/P from them was from 0.51 to 0.9, and only three of the pigs the result was doubtful. It should be noted that the commercial ELISA test (IDEXX) is designed to assess the level of antibodies in animals recover and therefore the value of S/P from 0.3 to 0.4 is questionable. Antibody titers against PVS were high and ranged from 1:512 to 1:2048. Unvaccinated animals remained seronegative.

The results of the control of infection showed that all vaccinated gilts, including indicators S/P 0,28; 0,34; 0,35 not get sick. In the process of monitoring, no clinical manifestations of disease were noted. Body temperature and vaccinated animals remained within the normal range.

Three control (unvaccinated) gilt sick with a typical clinical picture PRRS (depression, lack of appetite, conjunctivitis, cough, cyanosis of the skin) and increased body temperature.

Example 9.

To study the immunogenicity of the proposed vaccine, as well as determine its safety have been carried out experiments on two seronegative pregnant sows (47-50 days of gestation). Vaccine is injected once in the amount of 2 cm 3intramuscularly (behind the ear). 42 days after vaccination (90-93 day of gestation) sows were intranasally infected with virulent European PRRS virus, strain Lelystad, in the amount of 10 cm3vaccinated liquid with a titer of 4.0 lg TCC50/cm3. The animals were clinically observed for 4 months. After 37 days after vaccination were selected blood samples and serum were tested by ELISA (ARRIAH) for antibodies against PRRS virus. After vaccination and control of infection in pregnant sows were not noted any deviations from the physiological norm. The body temperature of the animals remained within normal values, appetite remained, animals reacted actively to the environment. Local reaction to the vaccine was absent.

37 days after vaccination in the blood serum of the animals was revealed specific antibodies to PRRS virus at titers of 1:400 and 1:200. These sows brought 16 piglets, 14 of them live healthy and 2 stillborn. Safety among the living healthy piglets during the suckling period was 100%.

In suckling piglets were sampled blood and serum tested for the presence of antibodies to PRRS virus. Before taking colostrum pigs remained seronegative, and 4-7 days after receiving colostrum in the blood serum of the new is born piglets revealed high titers of antibodies (1:400) against PRRS virus.

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

Example 10.

Study of the effectiveness of the proposed vaccine was conducted in a number of advantaged and disadvantaged by PRRS and PVIS pig farms.

To explore the tensions of post-vaccination immunity in sows vaccinated associated vaccine against PRRS and PVIS, was studied 5 pig farms. The vaccine was used according to the instruction for use of the associated emulsion inactivated vaccine against PRRS and PVIS, i.e. sows, gilts and boars-producers for the first time were vaccinated twice with an interval of 21 to 25 days for 2-3 weeks before insemination. Piglets obtained from vaccinated sows were inoculated in 1.5-2 months of age, twice with an interval of 20-30 days. From the vaccinated animals were taken blood samples 2-8 weeks after immunization, i.e. before insemination and in the first half of the pregnancy, during the second half of the pregnancy and immediately after farrowing. In addition, blood was collected from piglets obtained from these sows prior to receiving their colostrum and at different times after vaccination. Serum from animals investigated in the reactions of the NGA, ELISA (ARRIAH), rtha. The research results of blood sera from vaccinated pigs in the prosperous farms are presented in table 6.

From table 6 it is seen that in pigs of different age and sex groups through 55-70 days after vaccination serum samples revealed a high level of post-vaccination antibody. Significant differences in the strength of immunity in these groups of animals were noted.

The research results of blood sera from disadvantaged households from sows at different times of gestation and from piglets not receiving colostrum, is presented in table 7.

As can be seen from table 7, sows vaccinated associated vaccine against PRRS and PVIS, in serum samples revealed high titers of antibodies against the causative agents of these infections. After farrowing, the level of antibodies decreases slightly, but remains quite high. Thus, the proposed scheme immunization provides immunity against PRRS and PVIS at a high level for the entire period of gestation sows. The absence of antibodies in the blood sera "demoloshing" pigs is a direct measure of the effectiveness of vaccination.

Example 11.

Efficiency associated emulsion inactivated vaccine against PRRS and PVIS studied in a number of pig farms affected by PRRS and PVIS, in which the conductivity and epidemiological analysis before and after vaccination, given the reduction in the percentage of stillbirths and mortality of piglets. The results of the analysis of the epidemic situation in these farms are presented in table 8.

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

Thus, the associated emulsion inactivated vaccine against PRRS and PVIS is an effective drug for prophylaxis of these diseases, it is harmless and has sufficient immunogenicity. Despite the fact that the vaccine was applied in disadvantaged by PRRS and PVIS farms where there were a large number of sick animals, it is possible to improve the situation and within 5-24 months, the number of sows that brings the dead piglets decreased on average to 2.7%.

The above information shows the implementation of the use of the present invention, the following cumulative conditions:

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

for the present invention in the form as it Ohara is tirisano in the independent claim of the invention, confirmed the possibility of its implementation using the steps described in the application or known before the priority date tools and methods;

- when using the proposed vaccine against PRRS and PVIS is achieved technical result provided by the task of invention.

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

Sources of information

1. Wensvoort G. et. al. Mystery Swine Disease in the Netherlands: The isolation of Lelystad virus. - Vet.Quarterly, 1991, 13, 3, 121-130.

2. Mischenko V.A., Avilov, V.M. and other Reproductive and respiratory syndrome swine ("blue ear"). - Veterinary, 1994, No. 9, 22-24.

3. Syurin NR. and other Viral diseases of animals. - M.: UNITEMP, 1998, 552-558, 573-584.

4. PCT No. 92/21375; a 61 K 39/12, G 01 N 33/569 C 12 N 7/00; 10.12.92.

5. PCT No. 93/07898; a 61 K 39/12, G 01 N 33/569 C 12 N 7/00; 29.04.93.

6. Pat. France No. 2682966, C 12 N 7/02, 30.04.93.

7. EP 0676467; C 12 N 7/00, a 61 K 39/12, 12 P 21/08, From 07 To 16/10; 11.10.95.

8. Pat. U.S. No. 5587164, 424-218 .1, 24.12.96.

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11. Pat. UK No. 2282811; C 12 N 15/40, a 61 K 39/12, 07 K 14/18, C 12 N 7/02, 7/04; 19.04.95.

12. Orlyankin astray freight, Sergeev, V.A., and other Vaccine PVIS. - Veterinary medicine, 1989, No. 8, 28-30.

13. Grabec I., Erjavec I. and other Assessment antigenic activity of vaccines against parvovirus disease of pigs in laboratory animals. - Veterinary medicine, 1988, No. 11, 32-34.

14. Pat. Of the Russian Federation No. 1538305 And 61 To 39/295, 1.12.94.

15. Plana J., Vayreda M, Reu T. et.al. Results of challende in gilts vaccinated with a porcine vaccine against porcine parvovirus and Aujeszky''s disease. - Proc.8th. Inc. Pig.Vet.Soc.Congr. Ghent, Belgium, 1984, 11.

16. Application France No. 2781159 And 61 To 39/23, 21.03.2000.

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Table 1

Immunogenic activity of emulsion inactivated vaccine against PRRS and PVIS
No. of giltsThe level of antibodiesThe results of the control of infection by a PRRS virus through 35 days after vaccination
prior to vaccinationat 34 days post-vaccination
against PRRS (ELISA S/P)against PVIS (rtga)against PRRS (ELISA S/P)against PVIS (rtga)
10,041:640,551:2048 -
20,031:1280,901:2048-
30,071:1280,511:1024-
40,051:640,351:2048-
5(control)0,051:640,061:64+
6(control)0,061:640,071:64+
Note: "+" - clinical signs of disease;

"-" - absence of clinical signs of disease;

S/P is less than 0.3 - negative result;

S/P is equal to 0,3-0,4 - equivocal result;

S/P is greater than 0,4 - positive result.
Table 2

Immunogenic activity of emulsion inactivated vaccine against PRRS and PVIS
No. of giltsThe level of antibodiesThe results of the control of infection by a PRRS virus through 35 days after vaccination
prior to vaccinationat 34 days post-vaccination
about the Yves PRRS (ELISA, S/P)against PVIS (rtga)against PRRS (ELISA S/P)against PVIS (rtga)
10,011:640,651:1024-
20,081:320,401:2048-
30,011:320,971:1024-
40,021:640,401:2048-
5(control)0,021:640,061:64+
6(control)0,071:640,021:64+
Note: "+" - clinical signs of disease;

"-" - absence of clinical signs of disease;

S/P is less than 0.3 - negative result;

S/P is equal to 0,3-0,4 - equivocal result;

S/P is greater than 0,4 - positive result.

Table 3

Immunogenic activity of emulsion inactivated vaccine against PRRS and PVIS
No. of giltsThe level of antibodiesThe results of the control of infection by a PRRS virus through 35 days after vaccination
prior to vaccinationat 34 days post-vaccination
against PRRS (ELISA S/P)against PVIS (rtga)against PRRS (ELISA S/P)against PVIS (rtga)
10,081:320,401:2048-
20,041:320,301:2048-
30,051:320,971:1024-
40,021:321,201:2048-
5 (control)0,03 1:320,051:64+
6 (control)0,061:640,061:64+
Note: "+" - clinical signs of disease;

"-" - absence of clinical signs of disease.
Table 4

Indicators immunogenic activity associated vaccine against PRRS and PVIS in experiments on pigs and rabbits
No. of sample vaccineSod. AH, (%)AnimalsThe titers of antibodies in serum against virus
QtyPRRS (rnga)PVIS (rtga)
before WACC.30 days after vacc.before WACC.30 days after vacc.
143rabbits gilts5

3
<1:20 <1:20448±78 853±213 115±12

<1:32
512±0

1024±0
245rabbits gilts5

3
<1:20 <1:20480±101 426±10789±15

<1:32
614±102

1126±198
347rabbits gilts5

3
<1:20 <1:20512±78 533±10651±8

<1:32
1024±0

1365±341

Table 5

Indicators immunogenic emulsion inactivated vaccine against PRRS and PVIS on gilts
No. of giltsThe level of antibodiesThe results of the control of infection by a PRRS virus through 35 days after vaccination
prior to vaccinationat 35 days post-vaccination
against PRRS (ELISA)against PVIS (rtga)against PRRS (ELISA)against PVIS (rtga)
1 0,041:640,551:2048-
20,031:1280,901:2048-
30,071:1280,511:1024-
40,051:640,351:2048-
50,011:1280,481:1024-
60,011:1280,431:1024-
70,031:1280,341:1024-
80,051:640,671:512-
90,061:640,281:1024-
M±m0,04±0,01 99±180,50±0,061308±193
control
100,051:640,061:64+
110,061:640,071:64+
120,011:1280,021:128+
Note: the Value of S/P in ELISA≥0,4 - positive result;

"+" - the presence of the reaction temperature and the typical clinical manifestations;

"-" - absence of thermal reactions and clinical manifestations.
Table 6

The indicators of intensity of post-vaccination immunity in pigs of different age-sex groups grafted emulsion inactivated vaccine against PRRS and PVIS
The group of animalsQty animalsThe level of antibodies through 55-70 days against viruses
PRRS (ELISA)PVIS( rtga)
The main sows20217±293022±383
Repair mumps16294±323968±597
The grunts12208±433925±640
The piglets weaning24299±321653±262
Note: the value in the ELISA: <1:50 - negative result;

>1:100 positive result.

The value in rtga <1:64 - negative result;

>1:128 - positive result.

Table 7

The titers of antibodies in sows immunized against PRRS and PVIS
ManagementAge group of animalsNumber of samplesThe average level of antibodies against viruses
PRRS (rnga)PVIS (rtga)
sows:
before insemination19544±662157±342
JSC "Quiet flows the don"The 2nd floor. the gestation26586±532389±378
after farrowing10338±741706±264
piglets demolizione"10<1:20<1:32
sows:
before insemination18391±451678±263
PigThe 2nd floor. the gestation after farrowing17753±832078±305
"Kuznetsov"18271±501614±290
piglets demolizione"10<1:20<1:32
Sows:
before insemination11524±61 1914±387
JSCThe 2nd floor. the gestation13837±852934±503
"Ilinogorskii"after farrowing8500±701184±210
piglets demolizione"5<1:20<1:32
sows:
before insemination15395±542014±394
CJSC Nagaevo"The 2nd floor. the gestation17616±792409±328
after farrowing20238±691152±205
piglets demolizione"920±0<1:32
sows:
before insemination6350±801707±215
PigThe 2nd floor. the gestation after farrowing9675±821820±444
"Vlady is irski" 9568±471223±399
piglets demolizione"524±4<1:32

Table 8

Efficacy of emulsion inactivated vaccine against PRRS and PVIS in disadvantaged by these diseases farms
ManagementThe duration of vaccine (months)The level of reproductive pathology, %
before vaccinesafter applying vaccines
JSC "Wisniewski"2478,83,0
JSC "Quiet flows the don"2474,011,8
Pig Kuznetsovsky52*
CJSC Nagaevo"6702
Pig "lasarevskoye"2414,1*
APT Kirov1279,3*
JSC "Kommunar"666,6*
Note: * - single cases.

1. is aktina associated emulsion inactivated against reproductive-respiratory syndrome and parvovirus infection of pigs, containing antigenic material from the pathogen parvovirus infection of pigs reproduced in sensitive biological system and subjected to inactivation, and an oil adjuvant, characterized in that as antigenic material from the pathogen parvovirus infection of pigs it contains antigenic material from strain "VL-94-DEPT" swine parvovirus, collection VGNKI "VL-94-DEPT", and additionally antigenic material from strain "DB-DEPT" reproductive and respiratory syndrome swine collection VGNKI "DB-DEPT", in effective amounts.

2. The vaccine according to claim 1, characterized in that it contains antigenic material from strain "DB-DEPT virus reproductive and respiratory syndrome swine reproduced in transplantable cell culture Marc-145.

3. The vaccine according to claim 2, characterized in that it contains antigenic material from strain "DB-DEPT virus reproductive and respiratory syndrome swine reproduced in transplantable cell culture Marc-145 with biological activity, at least 6,0 lg TCD50/cm3.

4. The vaccine according to any one of claims 1 to 3, characterized in that it contains antigenic material from strain "DB-DEPT virus reproductive and respiratory syndrome swine reproduced in transplantable cell culture Marc-145 with biological activity, at least 6,0 lg TCD50/cm3 and inactivated by aminoethylethanolamine, the number 30,0-32,0 wt.%.

5. The vaccine according to claim 1, characterized in that it contains antigenic material from strain "VL-94-DEPT" swine parvovirus, reproduced in transplantable cell culture CPD.

6. The vaccine according to claim 5, characterized in that it contains antigenic material from strain "VL-94-DEPT" swine parvovirus, reproduced in transplantable cell culture CPD with a biological activity of at least 7,5 lg TCD50/cm3.

7. The vaccine according to claim 5 or 6, characterized in that it contains antigenic material from strain "VL-94-DEPT" swine parvovirus, reproduced in transplantable cell culture CPD with hemagglutinine activity of at least 1:2048 GUY.

8. The vaccine according to claim 7, characterized in that it contains antigenic material from strain "VL-94-DEPT" swine parvovirus, reproduced in transplantable cell culture CPD with a biological activity of at least 7,5 lg TCD50/cm3and HA activity of at least 1:2048 GUY and inactivated by aminoethylethanolamine, the number 15,0-17,0 wt.%.

9. The vaccine according to claim 1, characterized in that it contains antigenic material from strain "DB-DEPT virus reproductive and respiratory syndrome swine and antigenic material from strain "VL-94-DEPT" parvovirus pigs in a ratio of at least 2:1.

10. The vaccine according to claim 1, characterized in that from the oil adjuvants it contains oil adjuvant ARRIAH.

11. The vaccine of claim 10, characterized in that it contains an oil adjuvant ARRIAH in number at least to 53.0 wt.%.

12. The vaccine according to claim 1, characterized in that from the oil adjuvants it contains oil adjuvant "Montanide ISA-70".

13. The vaccine according to item 12, characterized in that from the oil adjuvants it contains oil adjuvant "Montanide ISA-70" in the amount of at least 53%.

14. The vaccine according to claim 1, characterized in that it contains antigenic material from strain "DB-DEPT virus reproductive and respiratory syndrome swine reproduced in transplantable cell culture Marc-145 with biological activity, at least 6,0 lg TCD50/cm3and inactivated by aminoethylethanolamine, antigenic material from strain "VL-94-DEPT" swine parvovirus, reproduced in transplantable cell culture CPD with a biological activity of at least 7,5 lg TCD50/cm3and hemagglutinine activity of at least 1:2048 GUY and inactivated by aminoethylethanolamine, and oil adjuvant at the ratio, wt.%:

Antigenic material from strain "DB-DEPT"
virus reproductive-respirato the aqueous syndrome pigs 30,0-32,0
Antigenic material from strain "VL-94-DEPT" swine parvovirus15,0-17,0
Oil adjuvantTo 100.0



 

Same patents:

FIELD: veterinary virology and biotechnology.

SUBSTANCE: invention proposes the strain that possesses high antigenic and immunogenic activity. The strain is deposited in the Collection of microorganism strain FGU VGNKI at the registration name "VNIIZZH-DEP". The coronavirus strain is reproduced in transplanted cell cultures of calf kidney (MDVK, Taurus), calve coronary vessel cells and in heterologous culture of simian cells (Vero). After 3-4 days of incubation at 37°C virus is accumulated in the amount up to 108 particels/cm3 by data of electronic microscopy. The strain shows ability for reproducing in the same accumulation degree in cell culture MDVK at 34°C. The strain possesses high biological, antigenic and immunogenic activity and retains native immunobiological properties after inactivation and can be used in preparing highly sensitive and specific diagnostic, highly immunogenic, harmless and areactogenic vaccine and curative preparations.

EFFECT: valuable properties of strain, improved preparing method.

6 tbl, 4 ex

FIELD: veterinary virology, biotechnology.

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

EFFECT: valuable veterinary properties of strain.

7 tbl, 8 ex

FIELD: veterinary virology, veterinary science, biotechnology.

SUBSTANCE: vaccine comprises antigenic material from the strain "VNIIZZH-DEP" of the cattle infectious rhinotracheitis virus reproduced in transplanted cells culture taken among transplanted cells BHK-21, MDBK and ZKG with the biological activity value at least 6.0 lg TCD50/cm3 and inactivated with aminoethylethyleneimine and also aluminum hydroxide as an adjuvant and, additionally, saponin taken in the effective ratios. The strain is deposited in the collection VGNKI at the registration code "VNIIZZH-DEP". Virus of the strain "VNIIZZH-DEP" is reproduced in transplanted cultures of cells BHK-21, MDBK and ZKG inducing cytopathic effect and virus is accumulated with the titer value 6.5-8.0 lg TCD50/cm3 for 48-96 h. Aminoethylethyleneimine is used in the concentration 0.1-0.2%, aluminum hydroxide is used as 3-4% gel in the amount 19.6-49.6 wt.-% and saponin is used as 10% aqueous solution in the amount 0.4 wt.-%. Vaccine shows high antigenic and immunogenic activity and harmless.

EFFECT: valuable properties of vaccine.

18 cl, 8 tbl, 8 ex

FIELD: biotechnology.

SUBSTANCE: claimed strain is obtained by hybridization of epidemic virus with cold adapted and temperature sensitive virus, which represents attenuation donor and is harmless for adults, and infants of 3-14 years old. Said virus makes it possible to obtain reassortant vaccine strains from new epidemic viruses. Strain is effectively cultivated in germinative hen embryos at 32°C, and has temperature sensitivity and cold adaptation. Reassortant has two genes derived from epidemic virus encoding surface proteins (hemagglutinin and neuroamidinase) and six genes derived from attenuation donor encoding non-glycosilated proteins. Strain has no reactogenicity in relation to adults and infants of 3-14 years old at intranasal application.

EFFECT: strain for living influenza intranasal vaccine with good biological properties and reactogenicity.

3 tbl, 2 ex

FIELD: veterinary virology and biotechnology.

SUBSTANCE: transmissible gastroenteritis of swine virus strain (TGSV) was isolated by sequential passages on SPEV (swine embryo kidney cell) and other cell cultures. Said virus strain is most effectively cultivated in 3-4-day monolayer of SPEV cells, obtained in roll-tube vessel, wherein after cultivation for 24-30 h infective activity level is 7.5 lg TCD50/cm3. Strain survives starting characteristics for 18-month storage and for 9-10 passages (observation time) on SPEV cell culture.

EFFECT: TGSV strain with high biological, antigenic and immunogenic activity useful in production of high-specific and sensitive diagnosticums and high-immunogenic and harmless inactivated vaccine preparations.

7 tbl, 5 ex

FIELD: veterinary virology and biotechnology.

SUBSTANCE: transmissible gastroenteritis of swine virus strain (TGSV) was isolated by sequential passages on SPEV (swine embryo kidney cell) and other cell cultures. Said virus strain is most effectively cultivated in 3-4-day monolayer of SPEV cells, obtained in roll-tube vessel, wherein after cultivation for 24-30 h infective activity level is 7.5 lg TCD50/cm3. Strain survives starting characteristics for 18-month storage and for 9-10 passages (observation time) on SPEV cell culture.

EFFECT: TGSV strain with high biological, antigenic and immunogenic activity useful in production of high-specific and sensitive diagnosticums and high-immunogenic and harmless inactivated vaccine preparations.

7 tbl, 5 ex

FIELD: veterinary virology and biotechnology.

SUBSTANCE: transmissible gastroenteritis of swine virus strain (TGSV) was isolated by sequential passages on SPEV (swine embryo kidney cell) and other cell cultures. Said virus strain is most effectively cultivated in 3-4-day monolayer of SPEV cells, obtained in roll-tube vessel, wherein after cultivation for 24-30 h infective activity level is 7.0-8.0 lg TCD50/cm3. Strain survives starting characteristics for 18-month storage and for 9-10 passages (observation time) on SPEV cell culture.

EFFECT: TGSV strain with high biological, antigenic and immunogenic activity useful in production of high-specific and sensitive diagnosticums and high-immunogenic and harmless inactivated vaccine preparations.

7 tbl, 5 ex

FIELD: virology, veterinary science.

SUBSTANCE: invention proposes a new strain of the avian reovirus ESASS № 99011475 belonging to a new antigen class of avian reoviruses. Also, invention proposes a method for preparing such reoviruses, a vaccine comprising such reovirus, a method for its preparing and a method for control against pathological states in poultries by using such vaccine. Proposed group of inventions provides enhancing the effectiveness of vaccination of poultries against reoviruses. Invention can be used in veterinary.

EFFECT: improved preparing method, valuable properties of strain and vaccine.

9 cl, 6 dwg, 3 tbl, 2 ex

FIELD: veterinary virology and biotechnology.

SUBSTANCE: invention relates to rotavirus strain having high biological, antigenic, and immune activity, as well as dominated properties in relation to epizootic isolates. Claimed virus strain is reproduced in single-layer passed cell cultures wherein after incubation for 24-48 h it accumulates in titer of 5.5-7.5 lg TCD30/cm3. (TCD - tissue cytopathogenic dose). Strain of present invention holds basic biological properties when passing in cell culture.

EFFECT: strain with increased biological, antigenic, and immune activity, inactivation resistance and dominated properties in relation to epizootic isolates.

12 tbl, 5 ex

FIELD: veterinary microbiology, virology, biotechnology.

SUBSTANCE: invention proposes vaccine that comprises as antigens the following components, vol. %: inactivated suspension of cells of hemolytic strains of microorganism M. bovis with the total concentration 100-120 billion cells in 1 cm3: strain M. bovis "G97-VNIVI", 4.0-5.0; strain M. bovis "SHZ-01", 4.0-5.0; inactivated cultural suspension of the strain Herpesvirus bovis type I "TKA-VIEV-V2" with infectious titer 107.0 - 107.5 TCD50/ml, 78.0-83.0; 6% solution of aluminum hydroxide gel, the balance. Vaccine enhances accumulation of specific protective antibodies in serum blood of vaccinated animals and provides the development of immunity against infectious keratoconjunctivitis that is retained for one year.

EFFECT: enhanced effectiveness and valuable properties of vaccine.

4 tbl, 6 ex

FIELD: veterinary microbiology, virology, biotechnology.

SUBSTANCE: invention proposes vaccine that comprises as antigens the following components, vol. %: inactivated suspension of cells of hemolytic strains of microorganism M. bovis with the total concentration 100-120 billion cells in 1 cm3: strain M. bovis "G97-VNIVI", 4.0-5.0; strain M. bovis "SHZ-01", 4.0-5.0; inactivated cultural suspension of the strain Herpesvirus bovis type I "TKA-VIEV-V2" with infectious titer 107.0 - 107.5 TCD50/ml, 78.0-83.0; 6% solution of aluminum hydroxide gel, the balance. Vaccine enhances accumulation of specific protective antibodies in serum blood of vaccinated animals and provides the development of immunity against infectious keratoconjunctivitis that is retained for one year.

EFFECT: enhanced effectiveness and valuable properties of vaccine.

4 tbl, 6 ex

FIELD: veterinary science, virology.

SUBSTANCE: invention proposes inactivated vaccine against pneumogastroenteritis in cattle and calves. Vaccine comprises a mixture of inactivated viruses of infectious rhinotracheitis, viral diarrhea, rota-, corona-, respiratory-syncytial infection and parainfluenza-3 of cattle and adjuvant. Suspensions of viruses in the mixture are taken in the ratio = 1:1:1:1:0.5:0.5, respectively, by volume. The titer value for viruses of infectious rhinotracheitis, viral diarrhea, rotaviral and coronoviral infection = 107.0 - 108.5 TCD50/ml and for viruses of parainfluenza-3 and respiratory-syncytial infection = 106.5 - 107.5 TCD50/ml. Also, invention proposes a method for prophylaxis of total pneumogastroenteritis in cattle and calves involving administrations of such vaccine. The proposed vaccine is more effective and shows enhanced immunogenicity that provides enhancing the prophylactic effect in its using. Invention can be used in animal husbandry.

EFFECT: improved, enhanced and valuable properties of vaccine.

2 cl, 4 tbl

The invention relates to biotechnology and associated with new vaccines

The invention relates to biotechnology and concerns associated vaccine containing a single drug multiple antigens
The invention relates to medicine, namely to Pediatrics, and can be used for vaccination against pertussis, diphtheria and tetanus

The invention relates to the field of medicine and relates to multivalent vaccine compositions containing acellular pertussis components of the vaccine, diphtheria toxoid, the tetanus toxoid and inactivated poliovirus

The invention relates to the field of veterinary biotechnology, Virology and Microbiology

The invention relates to veterinary medicine, Virology and biotechnology
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