RU2295567C1 - Strain of porcine reproductive and respiratory syndrome virus for production of diagnostic and/or vaccine preparations - Google Patents

Abstract

FIELD: virology.

SUBSTANCE: disclosed is strain of Porcine Reproductive and Respiratory Syndrome virus. Said strain is obtained by serial passages on 3-days Mare-145 cell culture. Said strain reproduces in cytoplasm of 3-days Mare-145 cell culture at 37°C. After 96 h incubation virus of 6-th passage is accumulated up to titer of 5.17lgTCD₅₀/ml.

EFFECT: strain with weak virulence for porcine and high biological, virulent and immunogenic activity in native form and after inactivation.

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Description

The invention relates to veterinary virology and biotechnology and can be used in the development and manufacture of diagnostic tools and / or specific prevention of pig reproductive and respiratory syndrome (PRRS).

PRRS is a contagious viral disease characterized by abortion at the end of gestation, the birth of a dead and weak offspring that dies in the first 2-3 days of life, and respiratory damage in piglets. The death of piglets can reach 80-90%.

The disease was first recorded and described in 1987 in the United States and Canada. The first outbreak of the disease with similar symptoms was noted in Germany in 1990, then this infection was recorded in most European countries.

In Russia, the disease was first noted in 1991 in the farms of the Kursk region. The disease spread rapidly and today PRRS is diagnosed in many regions and regions of Russia.

Currently, PRRS is widespread in many countries of the world with developed pig husbandry and often proceeds in enzootic (chronic) form (1-8).

The causative agent of the disease is an RNA-containing virus belonging to the genus Arterivirus, a family of Arteriviridae of the order Nidovirales.

For the first time, PRRS virus was isolated by Dutch scientists of the Central Veterinary Institute in 1991 in a culture of pig alveolar macrophage cells (9).

PRRSV strains isolated in different countries of Europe, North America and Asia are distinguished by virulence, antigenic activity, and the sequence of nucleotides in genomic RNA.

At least two PRRS virus genotypes are known - European and American. The homology between them at the nucleotide level is only 55-70%, and within the genotypes 97-99%.

The differences relate not only to variations in the nucleotide and amino acid structure, but also to the size of the viral proteins. So the size of the nucleocapsid protein of American strains of the PRRS virus is 123 amino acids, and the size of the nucleocapsid protein of European strains is 128 amino acids (10-12).

When conducting molecular characterization of domestic PRRS virus isolates, it was found that they belong to the European group, however, at the amino acid sequence level, differences of up to 16% are found, and the size of the nucleocapsid protein differs from that of both American (123 amino acids) and European strains (128 amino acids) and is 125 amino acids (13).

These structural features of domestic PRRS virus isolates concerning nucleocapsid as one of the most conserved viral proteins indicate their antigenic differences compared with the corresponding characteristics of West European and American virus isolates.

When comparing isolates of the PRRSV virus isolated on the territory of Russia, their extremely genetic and antigenic variability is found.

This circumstance forces us to constantly search for new PRRS virus isolates suitable for the manufacture of diagnostic and vaccine preparations.

Specific PRRS prophylaxis is carried out using live and inactivated vaccines. A number of foreign PRRS virus strains are known for use in the manufacture of diagnostic and vaccine preparations.

Known strain CNCM No. 1 - 1102 of the PRRS virus grown on sensitive hetero- or homologous cell cultures and used for the manufacture of diagnostic and vaccine preparations (14).

Known strain CNCM No. 1 - 1140 of the PRRS virus grown on sensitive hetero- or homologous cell cultures and used for the manufacture of diagnostic and vaccine preparations (15).

Known strain CNCM No. 1 - 1153 of the PRRS virus grown on sensitive hetero- or homologous cell cultures and used for the manufacture of diagnostic and vaccine preparations (16).

A known strain P120-117B of the PRRS virus obtained in homo- or heterologous cell cultures and used for the manufacture of diagnostic and vaccine preparations (17).

Known strains CNCM No. 1 - 1387 or CNCM No. 1 - 1388 of PRRS virus grown on sensitive hetero- or homologous cell cultures and used for the manufacture of diagnostic and vaccine preparations (18).

Known strain CNCM No. 1 - 1642 of the PRRS virus grown on sensitive hetero- or homologous cell cultures and used for the manufacture of diagnostic and vaccine preparations (19).

The known strain ATCC No. VR - 2332 of the PRRS virus grown in a green monkey kidney cell culture and used for the manufacture of diagnostic and vaccine preparations (20).

The known strain ATCC No. VR - 2402 of the PRRS virus grown in the culture of green monkey kidney cells and used for the manufacture of diagnostic and vaccine preparations (21).

A known strain of ATCC No. VR - 2509 of the PRRS virus, grown in CRL-12219 cell culture and used for the manufacture of diagnostic and vaccine preparations (22).

Known strain ATCC No. VR - 2525 of the PRRS virus grown on sensitive hetero- or homologous cell cultures and used for the manufacture of diagnostic and vaccine preparations (23).

PRAD virus strains NADC-8, NADC-9 and NVSL-14 are known to be grown in a sensitive biological system and used for the manufacture of vaccine preparations (24).

A known strain of JK-100 (CCTCC V20005) of the PRCC virus grown in a transplantable Marc-145 cell culture and used for the manufacture of diagnostic and vaccine preparations (25).

The known strain ECACC No. V-93070108 of the PRRS virus grown on sensitive hetero- or homologous cell cultures and used for the manufacture of diagnostic and vaccine preparations (26).

The disadvantages of the known strains are that they differ in their antigenic and immunobiological properties from the isolates of the PRRS virus epizootic virus isolated in Russia.

Of the isolates of the PRRS virus epizootic virus isolated in Russia, the “DB" strain of the homologous virus for the manufacture of diagnostic and vaccine preparations is known (27).

The disadvantages of this strain are its antigenic and immunobiological differences.

Closest to the proposed invention in terms of essential features is the strain CNCM No. 1-1102 of the PRRS virus grown on sensitive hetero- or homologous cell cultures and used for the manufacture of diagnostic and vaccine preparations (14).

The disadvantages of the prototype strain are its antigenic and immunobiological differences from isolates of the PRRS virus epizootic virus isolated on the territory of Russia.

The task of creating the present invention was to obtain a new production strain of PRRS virus with high biological, antigenic and immunogenic activity in its native form and after inactivation and suitable for the manufacture of sensitive and highly specific diagnostics and vaccines that create intense and prolonged immunity in vaccinated animals against circulating on the territory of Russia epizootic isolates of PRRS virus

The technical result from the use of the present invention is to expand the arsenal of production strains of PRRS virus with high biological, antigenic and immunogenic activity in its native form and after inactivation and suitable for the manufacture of sensitive and highly specific diagnosticum and vaccine preparations that create intense and prolonged immunity in vaccinated animals against Epizootic isolates of the PRRS virus circulating in Russia.

The specified technical result was achieved by obtaining the strain "CRC-96" (copyright) of PRRS virus for the manufacture of diagnostic and / or vaccine preparations.

The strain "KPR-96" is a new, previously unknown, in its native form, slightly virulent for pigs.

The original virus for obtaining the KPR-96 strain was isolated in 1996 from the blood serum of aborted sows with clinical signs of PRRS from the Victory Agricultural Union in the Leninsk-Kuznetsk district of the Kemerovo region, adapted by serial passages to transplanted kidney cell culture of the African green monkey Marc-145 and proposed as a manufacturing for the manufacture of diagnostic and / or vaccine preparations.

The strain "KPR-96" was deposited on October 28, 2004 in the All-Russian State Collection of strains of microorganisms used in veterinary medicine and livestock, Federal State Institution "All-Russian State Center for the Quality and Standardization of Medicines for Animals and Feed" under the registration number (link) "KPR- 96 "-DEP.

The strain "KPR-96" is weakly virulent for pigs and has high biological, antigenic and immunogenic activity in its native form and after inactivation. The possibility of its use for the manufacture of diagnostic and / or vaccine preparations has been experimentally confirmed.

The invention is illustrated in a graphical image that shows the results of comparing the amino acid sequences of the GP5 protein of strain "CRC-96" and strain Leiystad of the PRRS virus, as well as in the list of sequences in which: SEQ ID N0: 1 represents the nucleotide sequence of the gene ORS5 strain "CRC -96 "PRRS virus;

SEQ ID N0: 2 — amino acid sequence of the GP5 protein of the KPR-96 strain of PRRS virus.

The strain "CRC-96" PRRS virus is characterized by the following features and properties.

Morphological properties

The strain "CRC-96" of the PRRS virus belongs to the Arteriviridue family, the genus Arterivirus, is an RNA genomic, has morphological characteristics characteristic of the PRRS virus; the shape of the varion is spherical. The size of the virion is 45-75 nm with a nucleus occupying 3/4 of the varion with a diameter of 25-35 nm, surrounded by a lipid membrane.

Antigenic properties

The PRRS virus of the KPR-96 strain is stably neutralized by homologous antiserum.

The virus does not have hemagglutinating properties. When vaccinated, the antigen in strain "CRC-96" induces the formation of specific antibodies detected in the enzyme-linked immunosorbent assay (ELISA).

The antigenic activity and specificity of the PRRSV strain KPR-9A strain was determined by detecting specific antibodies in the serum of pigs obtained 28 days after their immunization with an inactivated emulsion vaccine made from PRRS virus strain KPR-96.

Prior to immunization in animals' blood sera, antibodies to PRRS virus were not detected, and 28 days after vaccination in all samples specific antibodies ranging from 1.79 to 2.05 were detected in ELISA.

Biotechnological characteristics

The strain "KPR-96" exhibits high biological activity. The biological activity of the strain was determined by titration on a Marc-145 cell culture.

Virus 6 and 17 passages obtained in a Marc-145 cell culture were used for titration after freezing-thawing twice and low speed centrifugation to precipitate cell detritus. The titration results on a Marc-145 cell culture showed that the biological activity of the CRC-96 strain of the PRRS virus of passage 6 averaged 5.17 lg TCD ₅₀ / ml, and passage 17 - 5.09 lg TCD ₅₀ / ml.

The difference in the titers of the virus 6 and 17 passages is not significant, i.e. as a result of 11 passages, the biological activity of the strain KPR-96 of the PRRS virus remained unchanged.

Chemo- and genotaxopomic characterization

The genome of the PRRS virus of the KPR-96 strain consists of a single molecule of linear positive single-stranded RNA about 15 thousand nucleotides in size. The 5'-end of the RNA is capped, and the 3'-end is polyadenylated. The capsid protein gene is located on the 3'-terminal part of the genome. The genome of the KPR-96 strain, like all other PRRS virus strains and isolates, contains eight open reading frames (ORFs) that encode renlicase genes (ORS 1a and 1b), envelope proteins (ORS 2-6), and nucleocapsid protein ( OPC 7). Six structural proteins of PRRSV virus and their corresponding genes were identified: a non-glycosylated nucleocapsid protein N (molecular weight 15 kDa) encoded by OPC 7, a non-glycosylated transmembrane protein M (molecular mass 18 kDa) encoded by OPC 6, and four N-glycosylated proteins with molar mass 25; 31-35; 45-50 and 29-30 kDa, encoded by OPC 5, OPC 4, OPC 3 and OPC 2, respectively. Spherical virions have a diameter of 45-75 nm, include a core with a diameter of 25-35 nm. The virion membrane contains lipids and carbohydrates as part of glycoproteins.

The identification and specificity of the PRRS virus was determined by polymerase chain reaction (PCR) and nucleotide sequencing.

As a result of the studies, only the PRRS virus genome was detected. The nucleotide sequencing of the OPC 5 gene confirmed that the strain "CRC-96" belongs to the European PRRS virus genotype. Comparison of the GP5 protein amino acid sequences derived from the OPC nucleotide sequences of the 5 amino acid sequences showed that the KPR-96 strain differs from the Lelystad strain in two amino acid residues (aa). Twentieth A.O. in “KPR-96” it is represented by phenylalanine, and in the strain Lelystad - by leucine, in position 37 of the GP5 protein in “KPR-96” and Lelystad are asparagine and aspartic acid, respectively.

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 / ml.

Resistance to external factors

The strain "KPR-96" is unstable to 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.

It is slightly virulent for pigs.

Oncogenicity is absent.

It is contagious in contact (the joint keeping of infected and healthy pigs).

Based on the data obtained, it can be argued that the strain “CRC-96" of the PRRS virus according to the antigenic and immunological spectra is an original and taxonomically new, previously unknown strain of the PRRS virus.

According to the applicant, the proposed strain meets the conditions of patentability "novelty" and "inventive step".

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

Example 1

In 1996, during a virological study of pig blood serum with clinical signs of PRRS obtained from the Victory agricultural complex of the Leninsk-Kuznetsk district of the Kemerovo region, the first PRRS virus isolate was isolated on transplanted Marc-145 cell culture. To isolate the PRRS virus from the field material, plastic culture mattresses with a monolayer of transplantable Marc-145 cell culture were used. A small volume (1 cm ³ ) of field blood serum from pigs with clinical signs of PRRS was infected with a Marc-145 cell culture and placed in a CO ₂ incubator for one hour at 37 ° C. After that, Needle medium with 10% content of fetal cattle of cattle and antibiotics was added to infected cells. Complete cytopathic effect (CPD) was apparent after 6 passages on a Marc-145 cell culture. Identification and specificity of the virus was determined in PCR with specificity control. The virus can also be isolated from the lungs, lymph nodes, spleen and other internal organs of infected piglets.

The isolated virus was used for mass infection of a 3-day culture of Marc-145 cells. Before the virus was introduced, the cell monolayer was washed once with phosphate-buffered saline (PBS). The culture was infected with a virus at a dose of 0.01-0.1 TCD ₅₀ per cell. Virus adsorption was carried out at 37 ° C for 1 hour. Every 10 minutes, the cells with the virus were shaken. After that, Needle medium was introduced into the material in a volume of 150-200 cm ³ with the addition of 5% fetal cattle serum, 50 μl / ml gentamicin and 0.3 mg / ml glutamine.

Cultivation was carried out at 36-37 ° C for 96-144 hours. As a control, 3 mattresses were left uninfected, in which the environment was changed.

Virus propagation in Marc-145 cell culture was determined by the characteristic CPD with the formation of a cluster of spherical cells rising above the monolayer. There should not be any destructive cell changes in the control mattresses. Mattresses in which CPD was observed with a lesion of 60% of the cell monolayer (usually after 48 hours) were selected and frozen at -20 ° C. The virus was obtained by freezing and thawing the infected cells twice, followed by removal of cell debris by centrifugation at 5000 rpm for 20 minutes. During 120 hours of incubation, passage 6 virus accumulated to a titer of 5.17 lg TCD ₅₀ / ml. The resulting strain of PRRSV virus (copyright name “KPR-96”) was deposited in the All-Russian State Collection of Microorganisms Used in Veterinary and Animal Husbandry of the Federal State Institution “All-Russian State Center for the Quality and Standardization of Medicines for Animals and Feed” (FGU VGNKI) October 28, 2004 under the registration number (link) "KPR-96" -DEP.

Example 2

The biological properties of the vaccine strain "CRC-96" (passage 6) were checked for the following indicators:

- lack of bacterial and fungal contamination;

- specificity and lack of viral contamination (PCR method);

- antigenic activity and specificity (ELISA);

- biological activity (titration on a Marc-145 cell culture);

- virulence for pigs.

1. Determination of the absence of bacterial and fungal contamination of the strain "KPR-96".

For this, Saburo media, meat-peptone broth (MPB), meat-peptone agar (MPA) and Kitta-Tarozzi medium were used. All bacterial media used were tested for growth properties according to GOST 28085-89. For testing of 3 vials (separately from each) with the culture virus, 1 cm ^{3 of} virus-containing material was introduced into 4 test tubes with thioglycolic medium. The tubes were incubated at different temperature conditions.

Two tubes with contents from each vial were kept in a thermostat at a temperature of 37 ± 0.5 ° C, one at 30 ± 0.5 ° C, and one at 22 ± 0.5 ° C. After 7 days, from 3 tubes incubated at 37 ± 0.5 ° C, reseeding was performed on the following bacterial media: agar and Saburo liquid medium, MPB and MPA, sugar broth and Kitta-Tarozzi medium. On Wednesday Kitta-Tarozzi was introduced in 1 cm ³ , and in the rest of the medium - 0.5 cm ^{3 of the} test material and kept for 7 days at a temperature of 37 ± 0.5 ° C, with Saburo medium - at a temperature of 22 ± 0, 5 ° C.

All tubes were subjected to daily visual inspection for 14 days.

The test results are presented in table 1. Tests showed that the strain “CRC-96" of the PRRS virus (passage 6) is not contaminated with bacterial and fungal microflora.

On all media with seeding and reseeding growth of bacterial and fungal microflora was not observed.

2. Testing the strain “CRC-96" of the PRRS virus for specificity and the absence of viral contamination by PCR.

Determination of specificity and the absence of viral contamination was carried out by examining a culture suspension of the PRRS virus strain KPR-96 by PCR for the presence of the genomes of PRRS viruses, CSF, Aujeszky's disease, PVIS, coronaviruses, circoviruses and pathogens of pig mycoplasmosis (M.hyopneumoniae, M .hyorhinis, M.hyosynoviae).

The weight of the study by PCR was carried out according to the "Guidelines for the indication of the PRRS virus genome by polymerase chain reaction", approved by the Department of Veterinary Medicine of the Ministry of Agriculture of the Russian Federation on February 21, 1997.

As a result of the studies, only the PRRS virus genome was detected in samples of the culture suspension. Genomes of CoES viruses, Aujeszky’s disease, PVIS, corona viruses, circoviruses and pathogens of pig mycoplasmosis (M.hyopneumonuie, M.hyorhinis. M.hyosynoviae) were not found.

Comparison of the complete nucleotide sequences of the nucleocanside protein gene (ORS 7) of the KPR-96 strain and other known strains of the PRRS virus showed a high degree of its homology with the Lelystad strain of the PRRS virus.

The level of nucleotide homology according to OPC 7 between strains "CRC-96" and "DB" is only 60%.

Significant differences between the KPR-96 and BD strains were also confirmed in ELISA (Ingenasa kit, Spain) by cross-sectional studies against the American and European types of PRRS viruses. The research results are presented in table 2. The data shown in table 2, indicate that the strain "CRC-96" belongs to the European gene group of the PRRS virus.

3. Determination of the specificity and lack of contamination of the strain "CRC-96" of PRRS virus in gilts.

For the test used 3 gilts weighing 25-30 kg. Weight animals were seronegative for PRRS viruses, PVIS, Aujeszky’s disease, CoES, porcine transmissible gastroenteritis (THES), influenza and M. hyopneumoniae.

All animals were injected intramuscularly with 5 ml of a culture suspension of the PRRS virus of the KPR-96 strain with a titer of 10 ⁵ ' ⁰ TCD ₅₀ / ml. Before and after 28 days after infection, blood samples were taken from the animals and serum was examined for the presence of antibodies from the following pathogens:

- PRRS virus in ELISA using a kit from IDEXX (USA), s / p value <0.4 - no specific antibodies, s / p≥0.4 - specific antibodies;

- PVIS virus in rtga using the “Set of drugs for serodiagnostics of PVIS in rtga” produced by FSI ARRIAH, value ≥1: 256 - the presence of specific antibodies;

- Aujeszky virus using a Chekit kit, s / p value <50 - no specific antibodies, s / p> 100 - specific antibodies;

- CoES virus using a Chekit kit, s / p-40 value - there are no specific antibodies, s / p> 50 - the presence of specific antibodies;

- THES virus in the microneutralization reaction, value ≥3.0 log ₂ - the presence of specific antibodies;

- M.hyopneumoniae using a Chekit kit, s / p value <20 - no specific antibodies; s / p> 30 - the presence of specific antibodies;

- swine flu virus using an IDEXX kit (USA), s / p <0.4 - no specific antibodies, s / p≥0.4 - the presence of specific antibodies.

The results of studies of blood serum for the presence of antibodies to the above pathogens are presented in table 3.

The data presented in table 3 show that in the tested samples antibodies to the viruses of PVIS, Aujeszky's disease, CoES, THES, influenza and M.hyopneumoniae were not detected. This indicates the absence of contamination of the strain "CRC-96" by the above pathogens.

4. Determination of antigenic activity and specificity of the strain "CRC-96" of the PRRS virus by ELISA.

These studies were carried out by ELISA by detecting specific antibodies in the serum of pigs obtained 28 days after immunization with an inactivated emulsion vaccine made from PRRS-96 strain KPR-96.

For this, 4 seronegative gilts weighing 25-30 kg were used. A virus with an infectivity titer of 5.0 lg TCD ₅₀ / ml was inactivated with aminoethylethyleneimine (AEEI) and emulsified with Montanide ISA-70 oil adjuvant in a 1: 3 ratio. After checking for sterility, the drug was administered to animals intramuscularly at a dose of 2 ml per head.

Blood samples were taken from all animals before and 28 days after vaccination. Blood serum was examined in a commercial ELISA kit from IDEXX (USA). The results of the study of the antigenic activity of the strain "CRC-96" of the PRRS virus by ELISA are presented in table 4.

The data in table 4 indicate that prior to immunization in serum gilts of antibodies to the PRRS virus were not detected, and after 28 days after vaccination in all 4 samples revealed specific antibodies ranging from 1.79 to 2.05 lg TCD ₅₀ / cm ³ .

Studies have also been conducted to study the antigenic activity of strains "DB", Lelystad and "CRC-96" of PRRS virus in pigs. The research results are presented in table 5.

From the data of table 5 it is clearly seen that the strain "CRC-96" has a pronounced antigenic activity.

After its introduction in blood serum of 2-3-month-old piglets, nonspecific antibodies to PRRS virus are detected at a sufficiently high level both with intranasal and intramuscular methods of introducing virus-containing material. In addition, the level of antibodies to PRRS virus 21 days after immunization with the strain "CRC-96" was higher than in piglets immunized with the strain "DB".

5. Determination of the biological activity of the strain "CRC-96" PRRS virus. The biological activity of the strain "CRC-96" was determined by titration on a culture of Marc-145 cells according to the standard method. Virus 6 and 17 passages grown in a Marc-145 cell culture were used for titration after freezing-thawing twice and low speed centrifugation to precipitate cell detritus.

A 2-3-day monolayer of Marc -145 cell culture grown in test tubes was used for titration of the PRRS virus. First, 10-fold dilutions (from 10 ^-1 to 10 ^-9 ) of virus-containing material were prepared on Eagle's medium without serum (pH 7.0-7.4). Each virus dilution was added to 4 cell culture tubes. Previously, growth medium was poured from the tubes, 0.1 cm ^{3 of the} diluted virus was added.

After 1 hour of contact, 0.9 cm ^{3 of} culture medium was added. On the tubes indicated the number of the test material (numerator) and dilution (denominator). The tubes were left in racks in an inclined position and placed in an incubator for incubation at a temperature of + 37 ° C.

At the same time, control was set with an uninfected cell culture. Every day for 6 days, titration results were recorded by viewing each tube under a small magnification of the microscope. After viewing, the PPD of the virus (conditionally in the crosses) was noted, the final results were taken in 144 hours after infection of the cell culture. The titration results were considered reliable while maintaining the monolayer and control.

The titer was calculated according to the Kerber method. Each passage of the test virus was titrated in 3 replicates. The results of determining the biological activity of the strain KPR-96 of the PRRS virus are presented in table 6. The titration results on the Marc-145 cell culture showed that the biological activity of the strain KPR-96 strain of the PRRS 6 virus is 5.17 lg TCD ₅₀ / ml, and passage 17 - 5.09 lg TCD ₅₀ / ml. The difference in the titers of the virus 6 and 17 passages is not significant, i.e. as a result of 11 passages, the biological activity of the strain KPR-96 of the PRRS virus remained unchanged.

Studies were also carried out of the cultural properties of the strains "DB", Lelystad, NVSL, No. 2156 and "CRC-96." The results of these studies are presented in table 7. From table 7 it is seen that the CPD of the strain “CRC-96” in the Marc-145 cell culture appears from 3 days after infection and reaches its maximum manifestation on 5 days, while the virus titer is 5.1 ± 0.07 lg TCD ₅₀ / ml.

6. Determination of the degree of virulence of the strain "CRC-96" PRRS virus in pigs.

The degree of virulence of the strain "CRC-96" for pigs was studied on pigs of 2-4 months of age with a live weight of 25-30 kg. The results of these experiments are presented in table 8.

From table 8 it is seen that a check of the strain “CRC-96” on pigs of 2-4 months of age at passage 17 showed that it is weakly virulence, since in some infected gilts only a slight increase in body temperature was observed (up to 40.7 ° C) within 1-4 days.

The resulting strain "CRC-96" was also tested on pregnant sows. Two seronegative pregnant pigs were intranasally infected 4 weeks before farrowing. Both sows were farrowed on day 114 after insemination. From them received 23 pigs. The characteristics of the offspring of sows are presented in table 9.

According to the data in table 9, the sows brought 18 living healthy piglets (78.3%) and 2 non-viable piglets (8.7%) and 3 with eye pathology (13%) were born in one sow. Two non-viable piglets died on the first day after birth. During the suction period, the remaining piglets, including those born with eye pathology, were not observed any clinical signs.

In the blood serum of piglets before receiving colostrum, antibodies to the PRRS virus were not detected. Examination of samples of the placenta of sows and internal organs of two forcedly killed piglets before receiving colostrum for the presence of the PRRS virus genome gave a negative result. At the same time, antibodies to the PRRS virus at a high level of s / p of 1.76 and 2.24 (in ELISA, IDEXX) were detected in the blood serum of sows taken on the day of farrowing.

A comparative study of the virulent properties of strains Lelystad, "DB" and "CRC-96" PRRS virus in pigs. The research results are presented in table 10.

From table 10 it can be seen that the clinical manifestation of PRRS in piglets infected with strains of "KPR-96", "DB" and Lelystad is significantly different, which indicates a different virulence of these strains. After infection of animals with strains of “KPR-96” and “DB”, it is seen that there are no clinical signs of the disease, with the exception of hyperthermia, which is typical for weakly virulent and avirulent strains of PRRS virus.

Thus, the test results showed that the strain "CRC-96" PRRS virus is weakly virulent.

Example 3

An inactivated emulsion vaccine against PRRS is prepared from the mother virus of the KPR-96 strain grown in a transplanted 3-day old Marc-145 cell culture with a concentration of more than 100,000 cells / ml. A mother virus is considered suitable for producing viral raw materials if it meets the following requirements: the infectivity titer after reproduction in a monolayer of Marc-145 cells is at least 5.0 lg TCD ₅₀ / ml, pH 7.2-7.4 and in the absence of contamination.

The virus is grown in mattresses with a capacity of 1.5 DM ³ with cell culture.

After washing off the growth medium and washing once with the FBI or medium, 5 ml of the 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, Needle medium is added to the mattresses in a volume of 150-200 ml with the addition of 5% fetal serum with an antibiotic (gentamicin at a concentration of 50 μg / ml or its analogues). Cultivation is carried out at 37 ° C for 72-120 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 characteristic CPD with the formation of a cluster 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 72-120 hours) are selected and frozen at -20 ° C.

The virus for the manufacture of the vaccine is obtained by 2-fold freezing and thawing by infection of the cells, followed by the discharge of the contents of the mattresses into one container, subject to sterile conditions. The resulting virus is freed from cell detritus by centrifugation at 1000 rpm for 20 minutes. The suspension purified from detritus should look like a clear pinkish-cherry liquid. The resulting virus is monitored for infectious activity.

A production series of the PRRS virus of the KPR-96 strain is considered suitable for the manufacture of vaccine preparations if it meets the following requirements: the infectivity titer after reproduction in a monolayer of Marc-145 cells is not less than 4.5 lg TCD ₅₀ / ml, pH 7.2-7 , 4 and in the absence of contamination. The purified virus-containing suspension is subjected to inactivation. Inactivation of the PRRS virus is carried out using a 1-2% aqueous solution of AEI, which is introduced into the virus-containing suspension to a final concentration of 0.05-0.08 wt.%. For this, an AEEI solution is added to the suspension heated to (26-28) ° C with constant stirring and the pH value is adjusted to 7.2-7.4 by adding a 5% succinic acid solution to the suspension. Inactivation of the virus is carried out in a thermostat at (374-2) ° C for 24 hours with periodic stirring. Upon completion of inactivation, the antigenic material is cooled to (4-6) ° C, its pH value is set in the range of 7.2-7.4, and antigen samples are taken to check for sterility and avirulence using methods known to the specialist.

An emulsion vaccine is prepared by dispersing a mixture of antigenic material and an oil adjuvant containing mineral oil with an emulsifier. As an oil adjuvant, a preparation of the company “Seppic” (France) under the brand name “Montanide ISA-70” is used.

The ratio of antigenic material from strain "CRC-96" of the PRRS virus and the Montanide ISA-70 oil adjuvant is 33% and 67%, respectively. Each vaccine dose of a 2 ml vaccine should contain 0.7 ml of PRRS virus antigen with a titer of at least 4.5 lg TCD ₅₀ / ml (before inactivation) and 1.3 ml of Montanide ISA-70 oil adjuvant.

The resulting vaccine is Packed in glass bottles and controlled in accordance with the technical conditions for sterility, safety and antigenic activity.

The vaccine was tested for sterility by plating on the following nutrient media: MPB, MPA, MPPB, Saburo medium and thioglycol medium. In all cases, microflora growth was not observed.

The safety of the vaccine was tested on six white mice and two gilts, which were injected subcutaneously and intramuscularly with 0.5 ml and 5.0 ml of the vaccine, respectively. The animals were monitored for 10 and 15 days, respectively, then they were euthanized and an autopsy was performed. During the observation period (10 days in mice and 15 days in gilts), all animals remained healthy. Slight swelling was observed at the injection site, and at the autopsy during examination of the injection site, single, small, encapsulated foci were observed with a diameter of 1-3 mm in mice and 5-7 mm in gilts, with a cut of which the vaccine contents were isolated.

Determination of the antigenic activity of the vaccine was carried out on six pigs 2.5-3 months old, seronegative with respect to PRSS, with a live weight of 25-30 kg. The vaccine was administered at a dose of 2 ml intramuscularly in the middle third of the neck (behind the ear) twice with an interval of 28 days. The animals were monitored for 45 days and blood samples were periodically taken. Blood serum was tested in ELISA using a commercial kit company IDEXX (USA) for the presence of specific antibodies to the PRRS virus. The results of the study of blood serum of piglets before and at various times after vaccination are presented in table 11.

According to table 11, specific antibodies to PRRS virus at s / p≥0.4 (positive values) in blood serum were detected by 21 days after vaccination in 5 animals out of 6 immunized.

28 days after vaccination, the average antibody level in the group significantly increased and amounted to s / p 0.95 ± 0.24, however, one animal remained serogenative. A week after revaccination, a significant increase in antibodies to the PRRS virus was observed, including that of a previously seronegative gilt, and its s / p index increased from 0.2 to 1.13. 14 days after revaccination, the average antibody level in the group was s / p 2.45 + 0.49. After revaccination, one of the piglets experienced a decrease in the level of antibodies from 0.5 to 0.35, which is probably due to the peculiarities of the immune system in this animal.

The results of clinical observation of vaccinated gilts showed that after immunization, no increase in body temperature at the injection site was recorded in all animals. The general condition of the animals remained within the physiological norm.

Thus, the resulting inactivated emulsion vaccine from the strain “KPR-96” is a sterile, harmless preparation and has sufficient antigenic activity. Double administration of the vaccine provides a high level of antibodies in the blood serum of the vaccinated animals.

Example 4

The effectiveness of the inactivated emulsion vaccine against PRRS from strain "CRC-96", made as described in example 3, was tested on pig farm No. 1 for 57,000 heads. Here, abortions were observed in sows, accompanied by stillbirth, as well as hollows and a high mortality rate in suckling pigs.

The reproductive livestock of the farm began to be inoculated with the associated emulsion inactivated vaccine against PRRS and PVIS produced by FSI ARRIAH, and piglets of the growing group were not vaccinated. Vaccination of the reproductive population has significantly reduced the number of abortions, the rate of stillbirth, free-flowing, as well as to increase the number and safety of offspring.

However, after this, the manifestation of respiratory syndrome in piglets of 45-50 days of age was noted.

Multiple studies of the material from sick animals carried out at FSI ARRIAH, using PCR, isolated the PRRS virus gene, as well as mycoplasmas (M. hyopneumoniae, M.hyorhinis), Haemophilis parasitis, Pasteurella multocida, Aclinobacillus pleuropneumoniae and type 2 circovirus.

To test the inactivated emulsion vaccine against PRRS from the strain "CRC-96" experimental and control groups were formed, each of which consisted of 3 sectors of 1000 heads, selected by type of analogues.

Pigs of the experimental group were vaccinated at 25 days of age with revaccination after 3-4 weeks at a dose of 2 ml per head, the control sectors were not vaccinated. All piglets were obtained from sows inoculated with the associated inactivated emulsion vaccine against PRRS and PVIS.

The animals of both groups were monitored throughout the growing period. After vaccination in the experimental group, thermometry was performed for 20 animals for 5 days, while the animal body temperature was within the normal range (38.4–40.1) ° С. A small swelling of 1.0 cm in diameter was felt at the injection site.

The test results of the emulsion inactivated vaccine against PRRS from strain "CRC-96" on weaned piglets in pig complex No. 1 are presented in table 12.

The data in table 12 indicate that in the experimental group (vaccinated sectors) during the growing period, the number of diseased animals was 8.1% less, forcibly killed less by 21.8%, 9.6% less were sent for sanitary rejects compared to with a control group. At the same time, the average daily gain was higher by an average of 3.5%.

The death in both groups was almost the same, but the overall safety of the animals during the rearing period in the experimental group was 3% higher.

An analysis of the incidence showed that in the experimental group, the majority of cases were animals with impaired gastrointestinal tract functions and injuries, and in the control group with respiratory disorders.

The results of the studies of blood serum of piglets in ELISA (ARRIAH), inoculated with an emulsion inactivated vaccine against PRRS from strain "CRC-96" in pig complex No. 1, are presented in table 13.

According to table 13, it follows that prior to vaccination, the percentage of seropositive animals for PRRS animals was 52.5%. This is due to the fact that at this time (24-28 days of life), colostral antibodies at a rather high level were detected in the blood serum of animals. This is due to the fact that pigs were obtained from sows who had been ill and vaccinated against PRRS and PVIS. After 26 days after vaccination, the number of seropositive animals decreased to 35%, which is associated with a sharp decrease in the level of colostral and insufficient formation of post-vaccination antibodies. After revaccination, the percentage of immune animals increased to 94.7%.

At the same time, the average antibody level increased from 66.1 ± 4.8 before vaccination to 82.2 ± 8.1 50 days after vaccination.

Example 5

The effectiveness of the inactivated emulsion vaccine against PRRS from strain "CRC-96", made as described in example 3, was tested on pig farm No. 2. In this farm, the disease manifested itself mainly in respiratory form on piglets of the rearing group at 45-55 days of age. The overall level of death and forced slaughter averaged 40-50%.

For research, experimental and control groups (sectors) were formed from piglets of 25-28 days of age. Animals were vaccinated at the age of 25-28 days of life, with revaccination after 3-4 weeks. At 35 days of age, these piglets were transferred to the rearing group, and weak and lagging animals were rejected. The test results of the vaccine from strain "CRC-96" on weaned piglets in pig complex No. 2 are presented in table 14.

From the data presented in table 14, it follows that in the experimental group, the incidence and mortality were lower than in unvaccinated animals. The average daily gain was also higher in the group of vaccinated animals (190 g versus 167 g in the control).

Safety over the growing period was also higher in vaccinated animals (68.1% versus 54.3% in the control).

The results of the studies of blood serum of piglets in ELISA (ARRIAH) obtained during the test vaccines from strain "CRC-96" in pig complex No. 2, are presented in table 15.

The data in table 15 indicate that before vaccination the percentage of animals seronegative in relation to PRRS was 52.5%.

This is due to the fact that at this time (24-28 days of life) colostral antibodies of a rather high level were detected in the blood serum of animals due to the fact that piglets were obtained from sows who had been ill and vaccinated against PRRS and PVIS. 26 days after vaccination, the number of seropositive animals decreased to 35%, which is associated with a sharp decrease in the level of colostral and insufficient formation of post-vaccination antibodies. After revaccination, the percentage of immune animals increased to 94.7%. At the same time, the average antibody level increased from 56.7 ± 8.0 before vaccination to 102.6 + 11.8 50 days after vaccination.

Example 6

The effectiveness of the inactivated emulsion vaccine against PRRS from strain "CRC-96", made as described in example 3, was tested on pig farm No. 2 for vaccination of the reproductive population. Sows and maintenance pigs were divided into two groups of 60 animals each. The animals of the experimental group were vaccinated with the test vaccine according to the temporary instructions for its use: the first time twice with an interval of 20-30 days three weeks before mating (insemination), then the animals were re-immunized for 60-70 days of gestation. In the control group, sows against PRRS were not vaccinated.

After immunization with the test vaccine in vaccinated animals, the total body temperature and the temperature at the injection site remained within the physiological norm; no clinical signs of the disease and deviations were noted.

The results of the test "KPR-96" on sows in pig farm No. 2 are presented in table 16.

From the data presented in table 16 it follows that in the experimental group, the yield of piglets is 1 sow higher than in the control (10.4 pigs versus 8.4). In vaccinated sows, the average weight of the newborn piglet was also higher compared to the control group (1.24 versus 1.21 kg).

During subsequent insemination (in the first hunt after the transfer of piglets for rearing) among sows previously vaccinated with emulsion inactivated vaccine from the KPR-96 strain, the sowing rate was lower than in the control group (6.6% versus 12% in control animals).

Example 7

The effectiveness of the inactivated emulsion vaccine against PRRS from strain "CRC-96", made as described in example 3, was tested on pig farm No. 3 for vaccination of the reproductive population. Sows and maintenance pigs were divided into two groups of 60 animals each. The animals of the experimental group were vaccinated with the test vaccine according to the temporary instructions for its use: the first time twice with an interval of 20-30 days three weeks before mating (insemination), then the animals were re-immunized for 60-70 days of gestation. In the control group, sows against PRRS were not vaccinated.

After immunization with the test vaccine in vaccinated animals, the total body temperature and the temperature at the injection site remained within the physiological norm; no clinical signs of the disease and deviations were noted.

As a result of vaccination, the productivity of sows in the experimental group became higher than in the control group (11 piglets per 1 sow in the experimental group versus 10 piglets in the control group).

In the experimental group, 31 pigs fell during the suction period, and in the control 35 piglets, i.e. the difference between the groups was 17.8%. The safety of sucking piglets in the experimental group was 96%, and in the control 94.1%.

Example 8

To prepare diagnostic serum for 6 seronegative gilts aged 4-6 weeks, the inactivated emulsion vaccine from the CRC-96 strain of PRRS virus is inoculated inoculum.

After 14 days, they are given the same vaccine intramuscularly. After 14 days, the animals receive hyperimmune serum and determine its activity in ELISA.

Example 9

To prepare the antigen for diagnostic purposes, the PRRS virus of the KPR-96 strain was inoculated into the Marc-145 cell culture.

After the appearance of the CPD, the supernatant is collected and concentrated 1000 times by ultracentrifugation, triton-100 is added to 0.2% concentration, centrifuged again for 10 minutes at 15,000 g. The supernatant is used as an antigen for diagnostic purposes. They do the same with the control (cell culture).

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

- strain "CRC-96" PRRS virus, embodying the invention, is intended for use in agriculture, namely in veterinary virology and biotechnology;

- for the proposed invention in the form 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;

- strain "CRC-96" PRRS virus obtained in accordance with the invention, has a high specific, immunogenic and antigenic activity, harmlessness and areactogenicity and expands the arsenal of tools suitable for the manufacture of diagnostic and / or vaccines against PRRS.

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

Information sources

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Table 1
The results of checking the viral preparation from the strain "CRC-96" for the absence of extraneous microflora No. of a bottle Thioglycol medium Reseeding on Wednesday MPA BCH Sah. b. K.-Tar. Ag. Saburo Wed Saburo 37 ° C 37 ° C 30 ° C 22 ° C 37 ° C 37 ° C 37 ° C 37 ° C 22 ° C 22 ° C one - - - - - - - - - - 2 - - - - - - - - - - 3 - - - - - - - - - -

Note: (-) - lack of growth of bacterial and fungal microflora on nutrient media.

table 2
ELISA study of pig serum for the presence of antibodies to PRRS virus Characterization of blood serum No. is alive. The level of antibodies to PRRS virus in ELISA, s / p IDEXX kit (for all types of virus) Ingenasa Set to the European type of PRRS virus to the american type of PRRS virus 21 days after immunization pcs. KPR-96 one 0.8 (+) 0.60 (+) 0.05 (-) 2 1.9 (+) 0.94 (+) 0.14 (-) 3 1.13 (+) 0.78 (+) 0.008 (-) 28 days after immunization KPR-96 one 1.55 (+) 0.96 (+) 0.32 (-) 2 1.93 (+) 1.07 (+) 0.63 (+) 3 1.27 (+) 1.03 (+) 0.36 (+) 28 days after immunization "DB" four 0.9 (+) 0.77 (+) 0.73 (1) 5 0.87 (+) 0.27 (-) 0.51 (+) 6 0.77 (+) 0.27 (-) 0.54 (+)

Note: “+” is a positive result;

"-" - negative result.

Table 3
The results of studies of pig serum for the presence of antibodies to pathogens PRRS, PVIS, Aujeszky’s disease, CoES, THES, M.hyopneumoniae and influenza Backlight Samples Pathogen serum test results PRRS PVIS Aujeszky CoES TGES M.hyo. flu one to imm. 0 1: 128 10 0 ≤2.0 3 0 28 days 1,5 1: 128 12 0 ≤2.0 four 0 2 to imm. 0 1:64 fourteen 0 ≤2.0 2 0 28 days 1,2 1:64 eighteen 0 ≤2.0 5 0 3 to imm. 0 1: 128 21 0 ≤2.0 3 0 28 days 1.8 1: 128 19 0 ≤2.0 6 0 Table 4
The results of determining the antigenic activity and specificity of the strain "CRC-96" of the PRRS virus by enzyme immunoassay Little pig The level of antibodies in ELISA, s / p before immunization after 28 days one 0 1.88 2 0 2.05 3 0 1.79 four 0 1.98

Note: ELISA IDEXX s / p <0.4 - specific antibodies are absent;

s / p≥0.4 - the presence of specific antibodies.

Table 5
Antigenic activity of strains "DB", Lelystad and "CRC-96" PRRS virus in pigs Virus strain Method and dose of infection No. alive. The level of antibodies in ELISA (IDEXX), s / p before immunization after immunization, days fourteen 21 Db Intramuscularly, 5 ml with a titer of 10 ^4.5 TCD ₅₀ / ml one 0.01 0.63 1,5 2 0.02 0.29 0, S 3 0.05 1,11 1.3 four 0.08 0.48 1,0 5 0,07 0.47 1.32 6 0.06 1,2 1.19 The average value, M ± S 0.05 ± 0.02 0.69 ± 0.37 1.2 ± 0.25 Lelystad Intranasally, 10 ml with a titer of 104.5 TCD ₅₀ / ml 7 0,03 0.6 0.98 8 0.05 0.4 0.78 9 0.01 0.46 0.46 The average value, M ± S 0.03 ± 0.02 0.49 ± 0.10 0.74 ± 0.21 KPR-96 Intranasally, 10 ml with a titer of 10 ^5.0 TCD ₅₀ / ml 10 0,07 0.44 1,5 eleven 0.08 0.31 1,2 12 0.09 0.6 1.8 The average value, M ± S 0.08 ± 0.01 0.45 ± 0.14 1.5 ± 0.24 Intramuscularly, 5 ml with a titer of 10 ^5.0 TCD ₅₀ / ml 13 0.01 N. and 2.6 fourteen 0,03 N. and 1.9 fifteen 0.04 N. and 2.2 The average value, M ± S 0.03 ± 0.01 N. and 2.2 ± 0.28

Note: NI - not investigated;

ELISA value (IDEXX kit)> 0.4 - positive serum;

<0.4 - negative serum.

Table 6
The results of determining the biological activity of the strain "CRC-96" virus PRRS Passage Experience number Virus breeding Virus titer lg TCD ₅₀ / ml 10 ^-2 10 ^-3 10 ^-4 10 ^-5 10 ^-6 10 ^-7 by group average value 6 one ++++ ++++ ++++ ++ - ---- - 5.0 5.17 ± 0.03 2 ++++ ++++ ++++ +++ - - - 5.25 3 ++++ ++++ ++++ +++ - - - 5.25 17 one ++++ ++++ ++++ ++ - - - 5,0 5.09 ± 0.07 2 ++++ ++++ ++++ ++ - - - 5,0 3 ++++ ++++ ++++ +++ - - - 5.25 cultural control one 2 3 Table 7
The cultural properties of certain strains of PRRS virus Virus strain The terms of the manifestation of the CPP, day The maximum manifestation of the CPP, day Virus titer, lg TCD ₅₀ / ml Db 2 3 7.0 ± 0.5 Lelystad four 5 4.5 ± 0.25 NVSL four 6 6.0 ± 0.25 No. 2156 four 6 3.25 ± 0.25 KPR-96 3 5 5.1 ± 0.07

Table 8
The results of the study of the virulence of the strain "CRC-96" in pigs Infection method (immunization) No. alive. Immunization result The level of specific antibodies in ELISA (IDEXX), s / p Clinical signs tamper. reaction before immunization in 21-28 days Intramuscularly 5 ml, titer 10 ^5.0 TCD ₅₀ one - - 0 2.6 2 - + 0 1.9 3 - + 0 2.2 Iitranasal 10 ml, titer 10 ^5.5 TCD ₅₀ four - - 0 1,5 5 - - 0 1,2 6 - + 0 1.8

Note: ELISA value (IDEXX kit)> 0.4 - positive serum;

<0.4 - negative serum.

Table 9
Characteristics of the offspring of sows infected with strain "CRC-96" virus PRRS Sow Piglets born Total living healthy unsustainable with eye pathology No. 1 13 8 2 3 Number 2 10 10 0 0 Total: 23 (100%) 18 (78.3%) 2 (8.7%) 3 (13%)

Table 10
Comparative study of the virulent properties of strains of Lelystad, "DB" and "CRC-96" PRRS virus in pigs Virus strain Method and dose of infection №№ Fever Wedge. signs day Maximum value Ts K.K. TO Leiystad intranasally, 10 ml with a titer of 10 ⁴ ' ⁵ TCD ₅₀ / ml one 2 42.0 + + + 2 2-3 41,4 + + + 3 one 41.2 + + - four 2 41.6 + + + 5 2-3 41.8 - + + 6 one 40.5 - + - "DB" intranasally, 10 ml with a titer of 10 ⁶ ' ⁵ TCD ₅₀ / ml 7 - - - - - 8 - - - - - 9 - - - - - 10 - - - - - eleven - - - - - intramuscularly, 5 ml with a titer of 10 ⁶ ' ⁵ TCD ₅₀ / ML 12 - - - - - 13 - - - - - fourteen - - - - - fifteen - - - - - 16 - - - - - KPR-96 intranasally, 10 ml with a titer of 10 ⁵ ' ⁰ TCD ₅₀ / ml 17 - - - - - eighteen - - - - - 19 - - - - intramuscularly, 5 ml with a titer of 10 ⁵ ' ⁰ TCD ₅₀ / ml twenty - - - - - 21 - - - - - 22 2-4 40.7 - - -

Note: C - cyanosis of the skin;

K.K. - conjunctivitis;

K - cough;

+ recorded a sign;

- did not observe the symptom.

Table 11
Antigenic activity of emulsion inactivated vaccine against PRRS from strain "CRC-96" on gilts No. is alive. The level of antibodies in ELISA ... days after immunization, s / p before immunization 21 28 * 7 (35) 14 (42) one 0.01 1,1 0.8 2.02 2.85 2 0.04 0.2 0.2 1.13 N. and 3 0,07 0.4 0.5 0.26 0.35 four 0.01 0.8 1.55 2,4 3.35 5 0.01 1.9 1.93 1.94 3.3 6 0.09 1.13 0.75 2,3 2,4 M + s 0.04 + 0.004 0.92 + 0.5 0.95 + 0.6 1.67 + 0.7 2.45 + 1.1

Note: * - re-immunization on day 28;

ELISA value (IDEXX) <0.4 - negative serum;

≥0.4 - positive serum;

N. and - not investigated.

Table 12
The test results of the emulsion inactivated vaccine against PRRS from strain "CRC-96" on weaned pigs in pig complex No. 1 №№ Indicators Experimental groups (vaccinated) Control groups (not vaccinated) The coefficient of change of the indicator,% (in experimental groups) one. Number of animals, goal. 1003 962 2. Age days 24-28 24-28 3. Transferred to growing goal. 1003 962 four. The average weight of 1 head, kg 7.3 ± 0.1 7.3 ± 0.1 5. Gross weight gain, kg 7326 7024 6. The average daily gain, g 216.6 ± 8.4 209.3 ± 10.4 + 3.5% 7. Ill, goal. % 250 261 24.9 27.1 -8.1% 8. Forced killed goal. % 43 53 4.3 5.5 -21.8% 9. Sanitary marriage, goal. % 103 110 10.3 11,4 -9.6% 10. It’s fallen, a goal. % 104 98 10.3 10,2 + 1.0% eleven. Transferred to fattening, goal. 753 701 12. Safety for the growing period. % 75.1 72.9 + 3.0%

Table 13
The results of studies of blood serum of piglets inoculated with an emulsion vaccine against PRRS from strain "CRC-96" in pig complex No. 1 Serum Sampling Dates Total blood serum samples The number of seropositive animals,% The average antibody level of positive samples,% inhibition investigated positive doubtful negative Before vaccination (age 24-28 days) twenty eighteen one one 52,5 66.1 ± 4.8 Before revaccination after 26 days. after vaccination (age 50-54 days) twenty four 3 13 35.0% 47.2 ± 9.7 50 days after revaccination (75-104 days of life) 19 eighteen 0 one 94.7 82.2 ± 8.1

Note: IFA value (ARRIAH) <20% - negative serum;

20-30% - doubtful serum;

≥ - 30% - positive serum

Table 14
The test results of the emulsion inactivated vaccine from strain "CRC-96" on weaned piglets in pig complex No. 2 №№ Indicators Experimental group (vaccinated) Control group (not vaccinated) one. Put on growing goal. 448 611 2. Ill, goal.% 155
34.6 289
47.3 3. Forced killed goal. % 106
23.7 138
22.6 four. It’s fallen, a goal. % 37
8.3 141
23.1 5. Transferred to sanitary machines, goal.% 122.7 10
1.6 6. The average daily gain, g 190 167 7. Transferred to fattening, goal.,% 293
65.4 322
52.7 8. Preservation for the period of growing,% 68.1 54.3

Claims (1)

The strain "KPR-96" virus of the reproductive and respiratory syndrome of pigs of the family Arteriviridae, genus Arterivirus, a collection of FSI VGNKI "KPR-96" - DEPT, for the manufacture of diagnostic and / or vaccine preparations.

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