RU2457859C1 - Cattle viral diarrhoeia vaccine - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to veterinary virology and biotechnology, and concerns a cattle viral diarrhoeia vaccine (CVDV). The described vaccine contains an avirulent and purified antigen material of the strain NADL-FGBI "Federal Centre for Animal Health" of the CVDV virus deposited in the collection of Federal State Institution Russian National Centre for Quality and Standartisation of Drugs and Feed for Animals (FGU VGNKI), No. NADL-FGBI "Federal Centre for Animal Health DEP prepared in the passaged mammal cell culture (the passaged cell cultures Taurus-2, PS and RBT) of infectious activity not less than 6,0 lg TCD₅₀/cm³ and ELISA activity not less than 6.6 log₂ to inactivation, in the effective amount, and target additives aluminium hydroxide (AHO) and saponin.

EFFECT: presented vaccine shows high antigenic and immunogenic activity and protects the immunised cattle livestock against the homologous CVDV agent.

13 cl, 9 tbl, 7 ex

Description

The invention relates to the field of veterinary virology and biotechnology and can be used in the development and manufacture of means for the specific prophylaxis of viral diarrhea (VD) in cattle.

VD (mucous membrane disease, mucosal disease, cattle infectious diarrhea, cattle infectious enteritis, newborn calf diarrhea) - acute cattle disease, mainly young animals, characterized by erosive-ulcerative inflammation of the mucous membranes of the digestive tract, rhinitis, enlarged lymph nodes, high fever oppression, leukopenia, constant or intermittent diarrhea, erosive and ulcerative stomatitis with profuse salivation, the appearance of mucopurulent discharge from the nasal strips and abortion, stillbirth, diarrhea, newborn calves and immunodeficiency.

VD cattle is one of the most common viral infections. The disease is registered in many countries of the world, including in Russia. The disease causes great economic damage to livestock. Losses to livestock farms from cattle VD are the result of mortality and forced slaughter of young animals, loss of fatness, decreased milk productivity, abortion, the birth of non-viable calves, etc.

VD is most often manifested in the form of epizootic outbreaks among young animals on livestock farms and complexes. Morbidity ranges from 10 to 100%, mortality - from 5 to 95%. It depends on the virulence of the strain of the virus, the resistance of the animal organism, the level of immunity, animal health conditions.

The main source and reservoir of the causative agent of VD of cattle are persistently infected animals, which, when the organism is clinically healthy, secrete a virus, infecting susceptible cattle.

Vaccine prophylaxis of VD takes a leading place in the complex of antiepizootic measures aimed at combating this disease.

For specific prophylaxis of VD of cattle, live and inactivated vaccines are used.

Live vaccines are currently widely tested with good results, but they have several disadvantages. Due to the presence of reactogenicity, they are not recommended for vaccination of pregnant animals, since during intrauterine transmission the virus can enter the fetus, which is naturally more susceptible to infection, which can result in an abortion or the birth of a persistently infected calf. Live vaccines have a certain immunosuppressive effect, which can lead to the activation of other agents of a bacterial and viral nature. In addition, the vaccine virus can persist for a considerable time in the body of vaccinated animals, which leads to a wide spread of the virus in the external environment, creates the danger of its reversal and prevents the implementation of diagnostic measures. In addition, attenuated strains of the causative agent of VD of cattle induce, as a rule, the state of immunosuppression in pregnant cows and persistently infected calves. For this reason, in the post-vaccination period in animals, an increase in the frequency of second, sometimes lethal infections is noted.

Along with virus vaccines against VD of cattle, inactivated vaccines, both sorbed and emulsion, are widely used (1-6).

Known vaccine against VD of cattle adsorbed inactivated, containing the active substance in the form of avirulent and purified antigenic material from the Oregon C-24V strain of VD virus of cattle, obtained in a sensitive biological system, and the target additive in the form of an aluminum hydroxide (GOA) adjuvant sorbent in effective ratio (1).

Known vaccine against VD of cattle inactivated adsorbed, containing the active substance in the form of avirulent and purified antigenic material from the Singer strain of VD virus of cattle, obtained in a sensitive biological system, and the target additive in the form of an adjuvant-sorbent GOA in an effective ratio (1).

Known vaccine against VD of cattle inactivated sorbed, containing the active substance in the form of avirulent and purified antigenic material from strain "New York-1" virus of VD cattle, obtained in a sensitive biological system, and the target additive in the form of an adjuvant-sorbent GOA in an effective ratio ( one).

Known vaccine against VD of cattle inactivated sorbed, containing the active substance in the form of avirulent and purified antigenic material from the Kazakhstan strain of the VD virus of cattle, obtained in a sensitive biological system with an infectious activity of 5.5 ÷ 6.0 lg TCD ₅₀ / cm ³ before inactivation , and the target additive in the form of an adjuvant-sorbent GOA in an effective ratio (7).

The closest to the invention according to the set of essential features is an inactivated adsorbed vaccine against VD of cattle, containing the active substance in the form of avirulent and purified antigenic material from the Oregon C-24V strain of VD virus of cattle obtained in a monolayer transplanted culture of KST cells with an infectious activity of at least 7.0 lg TCD ₅₀ / cm ³ before inactivation, and the target additive in the form of an adjuvant-sorbent GOA in an effective ratio (8, 9).

The disadvantages of the known vaccines inactivated adsorbed against VD of cattle, including the vaccine prototype, are that they provide effective protection of animals only from infection with a homologous virus.

The problem to which the present invention is directed, is to expand the arsenal of vaccines against VD of cattle inactivated adsorbed, obtained on the basis of new production strains of the virus of VD cattle with high biological, antigenic and immunogenic activity in their native form, preserving antigenic and immunogenic activity after inactivation and suitable for the manufacture of highly immunogenic, harmless and areactogenic vaccine preparations that provide effective protection for susceptible animals x from the homologous causative agent of VD cattle.

The technical result from the use of the present invention is to expand the arsenal of highly immunogenic, harmless and areactogenic vaccines against VD of cattle adsorbed inactivated, able to protect the livestock of cattle from the homologous pathogen of VD of cattle.

The specified technical result was achieved by creating a vaccine against VD of cattle adsorbed inactivated, characterized by the following set of features.

The proposed vaccine contains the active substance in the form of avirulent and purified antigenic material from the strain "NADL-ARRIAH" (author's name) virus VD cattle obtained in a sensitive biological system, target additives in the form of an adjuvant-sorbent GOA and adjuvant saponin in an effective ratio.

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

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

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

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

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

To obtain antigenic material from the strain “NADL-ARRIAH” virus VD cattle as a sensitive biological system using mainly transplantable cell culture of Taurus-2.

If necessary, transplantable PS and RBT cell cultures can also be used to obtain antigenic material from the NADL-ARRIAH strain of the VD virus of cattle.

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

To inactivate the virus, aminoethylethyleneimine (AEEI) is used, which is added to the purified virus-containing suspension to a concentration (%): 0.1 ÷ 0.2. Inactivation of the virus is carried out at 37 ° C for 24 hours. At the end of the inactivation, AEEI is neutralized by adding sodium thiosulfate to the antigen suspension.

The proposed vaccine contains avirulent and purified antigenic material from the strain "NADL-ARRIAH" virus VD cattle, obtained mainly in transplantable cell culture of Taurus-2 with an infectious activity of not less than 6.0 lg TCD ₅₀ / cm ³ and activity in an enzyme-linked immunosorbent assay (ELISA ) not less than 6.6 log ₂ before inactivation, in the amount of (wt.%): 50.0 ÷ 80.0.

If necessary, the proposed vaccine may contain avirulent and purified antigenic material from the strain "NADL-ARRIAH" virus VD cattle obtained in transplantable culture of RBT cells with an infectious activity of not less than 6.0 lg TCD ₅₀ / cm ³ and activity in ELISA not less than 6, 6 log ₂ before inactivation, in the amount (wt.%): 50.0 ÷ 80.0.

If necessary, the proposed vaccine may contain avirulent and purified antigenic material from the strain "NADL-ARRIAH" virus VD cattle obtained in transplantable culture of PS cells with an infectious activity of not less than 6.0 lg TCD ₅₀ / cm ³ and activity in ELISA not less than 6, 6 log ₂ before inactivation, in the amount (wt.%): 50.0 ÷ 80.0.

As a target additive, the proposed vaccine contains an adjuvant-sorbent GOA in the amount (wt.%): 19.6 ÷ 49.6.

If necessary, as a target additive, the proposed vaccine may additionally contain adjuvant saponin in an amount of 0.4 wt.%.

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

1. VD vaccine cattle adsorbed inactivated.

2. The active substance in the form of avirulent and purified antigenic material from the strain "NADL-ARRIAH" virus VD cattle in an effective amount.

3. Targeted supplements.

The signs of the invention, characterizing the proposed vaccine and coinciding with the signs of the prototype, including the generic concept, reflecting the purpose, are:

1. VD vaccine cattle adsorbed inactivated.

2. The active substance.

3. Targeted supplements.

Compared with the prototype vaccine, an essential distinguishing feature of the proposed vaccine is that it contains an avirulent and purified antigenic material from the strain "NADL-ARRIAH" virus VD cattle in an effective amount.

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

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

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

3. Avirulent and purified antigenic material from the strain "NADL-ARRIAH" virus VD cattle obtained in transplantable cell culture of Taurus-2 with an infectious activity of at least 6.0 lg TCD ₅₀ / cm ³ and activity in ELISA of at least 6.6 log ₂ before inactivation, in the amount (wt.%): 50.0 ÷ 80.0.

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

5. Avirulent and purified antigenic material from the strain "NADL-ARRIAH" virus VD cattle obtained in transplantable cell culture PS with an infectious activity of not less than 6.0 lg TCD ₅₀ / cm ³ and activity in ELISA of not less than 6.6 log ₂ to inactivation, in quantity (wt.%): 50.0 ÷ 80.0.

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

7. Avirulent and purified antigenic material from the strain "NADL-ARRIAH" virus VD cattle obtained in transplantable culture of RBT cells with an infectious activity of not less than 6.0 lg TCD ₅₀ / cm ³ and activity in ELISA of not less than 6.6 log ₂ to inactivation, in quantity (wt.%): 50.0 ÷ 80.0.

8. Of the target additives adjuvant sorbent GOA.

9. GOA in the amount (wt.%): 19.6 ÷ 49.6.

10. Of the target additives, an additional adjuvant is saponin.

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

12. Avirulent and purified antigenic material from the strain “NADL-ARRIAH” of the VD virus of cattle obtained in a transplantable mammalian cell culture taken from the group comprising Taurus-2 cell culture, PS cell culture and RBT cell culture, with an infectious activity of at least 6 , 0 lg TCD ₅₀ / cm ³ and activity in ELISA not less than 6.6 log ₂ before inactivation, and target additives GOA and saponin in the ratio, wt.%:

Antigenic material 50.0 ÷ 80.0 GOA 19.6 ÷ 49.6 Saponin 0.4

The proposed vaccine expands the arsenal of highly immunogenic, harmless and areactogenic vaccines against VD of cattle adsorbed inactivated, which provide effective protection of the cattle population from infection by a homologous causative agent of VD of cattle.

The achievement of the technical result from the use of the invention is explained by the fact that the vaccine contains avirulent and purified antigenic material from the new strain NADL-ARRIAH of the VD virus of cattle, reproduced in Taurus-2, PS and RBT cell cultures with high infectious activity before inactivation and having high antigenic activity and immunogenicity after inactivation.

The possibility of its use for the manufacture of a vaccine against VD of cattle adsorbed inactivated, which provides effective protection of cattle against a homologous pathogen, has been experimentally confirmed.

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

Morphological features

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

Antigenic properties

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

The virus is stably neutralized by homologous antiserum.

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

Genotaxonomic characteristic

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

10 proteins were isolated and characterized: 4 structural and 6 non-structural. Single-stranded RNA is bound to the p14 / C capsid protein surrounded by external glycoproteins (gp25 / E1, gp53 / E2, gp48 / E0) and the lipid membrane.

External glycoprotein gp53 / E2 induces the production of virus-neutralizing antibodies in high titers in animals. The gp48 / E0 protein is able to induce a high level of antibodies in infected animals, but with low virus-neutralizing activity. In infected animals, antibodies to gp25 / E1 are produced in low titers.

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

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

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

Biotechnological characteristic

The strain “NADL-ARRIAH” of the VD virus of cattle is reproduced in transplanted cultures of calf kidney cells (MDBK, Taurus-2, PT, RBT), transplanted cell culture line PS, transplanted cattle intestinal mucosal cell line culture (FBI) and intraspecific hybrid transplanted culture line kidney cells of a pig embryo with pig splenocides (A ₄ C ₂ ).

At 37 ° C for 72–96 hours of incubation, the virus accumulates in titers of 6.0–7.0 lg TCD ₅₀ / cm ³ . The strain "NADL-ARRIAH" virus VD cattle is stable and retains its properties for 10 passages (observation period).

Physical properties

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

Resistance to external factors

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

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

Lyophilization has almost no effect on its activity, however, repeated freezing and thawing reduce the virulence and immunogenicity of the virus.

Additional features and properties

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

Reactogenicity - does not possess reactogenic properties.

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

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

Virulence - expressed.

Contagiousness - expressed.

Oncogenicity is absent.

Free from contamination by bacteria, mycoplasmas and extraneous viruses.

Based on the data obtained, it can be argued that the strain "NADL-ARRIAH" according to antigenic and immunological spectra is the original strain of the virus VD cattle.

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

Example 1

The source for the production of the NADL-ARRIAH virus strain VD of cattle was the reference strain NADL of the VD virus of cattle, obtained by the Federal State Institution ARRIAH on August 11, 2004 from the collection of typical ATCC USA cultures. The vaccine strain was obtained by FGU "ARRIAH" by culturing Taurus-2, RBT and PS cells in transplantable cultures.

The results of studies on the adaptation of the VD virus of cattle of the strain "NADL-ARRIAH" to transplantable cell cultures of Taurus-2, RBT and PS are presented in tables 1, 2 and 3.

The resulting virus was subjected to comprehensive control in accordance with the OIE guidelines for standard diagnostic methods and vaccines (1996) and at the level of the eighth passage was deposited as a mother seed. The seed brood virus with an infectious activity titer of 6.5 ÷ 7.0 lg TCD ₅₀ / cm ³ is presented as a lyophilized culture fluid and stored at a temperature of minus 40 ° С.

The resulting strain of the virus VD cattle assigned the copyright name "NADL-ARRIAH".

Example 2

To obtain a vaccine against VD of cattle, the adsorbed inactivated matte virus of the NADL-ARRIAH strain is reproduced by infection of a monolayer of a transplanted Taurus-2 or RBT cell culture or PS grown in 1.5 dm ³ mattresses or 3 dm ³ rotating vessels. Cooled at 4 ° C, the virus-containing suspension, subject to sterile conditions, is freed from cellular detritus in a known manner using low-speed centrifugation (2500 ÷ 3000 rpm for 30 minutes) and poured into a container.

The suspension freed from detritus should look like a clear pink liquid. Then, a sample is taken from the container for the production control of viral raw materials. The production series of the VD virus of cattle should have an infectious activity of not less than 6.0 lg TCD ₅₀ / cm ³ and activity in ELISA not less than 6.6 log ₂ .

Inactivation of viral raw materials is carried out using a 6% AEI solution introduced into the viral suspension to a final concentration of 0.1 ÷ 0.2%. To do this, in the suspension, heated to 26-28 ° C, the AEEI solution is added with constant stirring and the pH of the suspension is set within 7.6–7.8 with a 5M solution of acetic acid. Inactivation of the virus is carried out at a temperature of (37.0 ± 0.5) ° C for 24 hours with periodic stirring of the suspension (for 3 ÷ 5 minutes every 5 ÷ 6 hours).

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

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

The resulting antigenic material is used for the manufacture of the sorbed form of the vaccine against VD of cattle.

As an adjuvant sorbent in the proposed vaccine, a sterile 3 ÷ 4% GOA gel is used.

The concentration and volume of GOA in the final preparation and graft dose are determined according to the well-known formula depending on the titer of the virus in the initial suspension. The optimal amount of GOA in the vaccine can be (wt.%): 19.6 ÷ 49.6. The mixture of the suspension with GOA is stirred for 1 hour and left for sedimentation. Then an additional 10% aqueous solution of saponin adjuvant is added to the vaccine semi-finished product at the rate of 2.0 mg per vaccine dose, bringing the concentration of saponin in the drug to 0.4 wt.%. The mixture before packaging is kept at a temperature of 2 ÷ 6 ° C for 120 hours with periodic stirring.

The optimal component composition (wt.%) Of the obtained vaccine against VD of cattle is shown in table 4.

The content of antigen in the vaccine in the above range is its effective amount in the drug, ensuring the achievement of a technical result.

The resulting vaccine is a pink liquid with a loose sorbent precipitate, which, when shaken, easily breaks into a homogeneous suspension.

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

The sterility of the vaccine is determined in accordance with GOST 28085. The virulence of the drug is determined by the method of three consecutive passages of inactivated antigen in transplanted cell cultures by the absence of CPD.

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

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

The antigenicity of each vaccine series is tested in rabbits. To do this, take 7 rabbits weighing 2.0 ÷ 2.5 kg. The vaccine is administered to five rabbits subcutaneously in the back at 2 cm ³ . Before vaccination and 14 days after the double injection of the vaccine with an interval of 20 ÷ 25 days, the rabbits are bled, kept at 37 ° C, the serum is separated and examined in the ELISA for the presence of antibodies to the VD virus of cattle.

To set up a reaction, 0.1 cm ^{3 of a} solution of a specific viral antigen in 0.05 M carbonate-bicarbonate buffer (pH 9.5 ÷ 9.6) in working dilution is added to the wells of plates and incubated for 18-20 hours at 4, 0 ° C. By shaking, the unbound contents of the plate wells are removed and washed 3 times with wash buffer.

In sensitized tablets make blocking buffer in the amount of 0.05 cm ³ per well. Incubated for 1 hour at a temperature of 37.0 ° C in a static state. Wash 3 times with wash buffer.

0.1 cm ^{3 of the} test and control sera in a 1:50 dilution are added to the horizontal row and a series of double dilutions of the test and control sera from 1:50 to 1: 6400 is prepared. Incubated at (37.0 ± 0.5) ° C for 1 hour and washed 3 times with wash buffer.

0.05 cm ^{3 of} anti-rabbit IgG conjugate in working dilution was added to the washed wells, incubated for 1 h at 37.0 ° C, and the wells were washed with the same solution.

Then, 0.05 cm ^{3 of the} prepared substrate - chromatogen (ABTS) solution is added to each well. Incubated for 15 ÷ 20 min at room temperature, the reaction is stopped with 1% SDS solution (0.05 cm ³ ).

The reaction is taken into account using a spectrophotometer, recording the optical density (OD) at a wavelength of 405 nm.

Determine the average OD value for the wells with control negative serum. Serum titer is considered its maximum dilution, the optical density in which is 2 times higher than the optical density in wells with a control negative serum.

The vaccine series is considered to have passed the control for antigenic activity if the titer of specific antibodies to the VD virus of cattle in ELISA is not lower than 1:50.

From the obtained antigenic material, 4 series of the vaccine against VD of cattle adsorbed inactivated, differing in the content of active substance and adjuvant of GOA sorbent in the preparation, were prepared.

Series No. 1 of the proposed vaccine contains, wt.%:

Avirulent and purified antigenic material from the strain "NADL-ARRIAH" VD virus of cattle obtained in transplantable Taurus-2 cell culture with infectious activity 6.5 lg TCD ₅₀ / cm ³ and ELISA activity 6.6 log ₂ before inactivation (hereinafter “Antigenic material) 50,0 GOA 49.6 Saponin 0.4

Series No. 2 of the proposed vaccine contains, wt.%:

Antigenic material 70.0 GOA 29.6 Saponin 0.4

Series No. 3 of the proposed vaccine contains, wt.%:

Antigenic material 80.0 GOA 19.6 Saponin 0.4

Series No. 4 of the proposed vaccine contains, wt.%:

Antigenic material 60.0 GOA 39.6 Saponin 0.4

Example 3

Tests of antigenic activity of series No. 1 of the proposed vaccine against VD of cattle, manufactured as described in example 2, and containing, wt.%:

Antigenic material 50,0 GOA 49.6 Saponin 0.4

The antigenic activity of the obtained preparation was determined on guinea pigs weighing 0.45 ÷ 0.50 kg.

The research results are presented in table 5. According to the data of table 5 it is seen that the vaccine against cattle VD adsorbed inactivated from the strain "NADL-ARRIAH" has a high antigenic activity.

Example 4

Tests of antigenic activity of series No. 2 of the proposed vaccine against VD of cattle, made as described in example 2, and containing, wt.%:

Antigenic material 70.0 GOA 29.6 Saponin 0.4

The antigenic activity of the obtained preparation was determined on rabbits weighing 2.0 ÷ 2.5 kg.

The research results are presented in table 6. According to the data in table 6 it is seen that the vaccine against cattle VD inactivated adsorbed from the strain "NADL-ARRIAH" has a high antigenic activity.

Example 5

Tests of antigenic activity of series No. 3 of the proposed vaccine against VD of cattle, made as described in example 2, and containing, wt.%:

Antigenic material 80.0 GOA 19.6 Saponin 0.4

In order to study the antigenic activity of the vaccine against VD of cattle adsorbed inactivated from the strain “NADL-ARRIAH” in the production environment, it was used in a non-VD cattle farm of OJSC PSH “Luchinskoe” in the Sobinsky district of the Vladimir region to prevent this disease. Before vaccination, mass diseases of calves 1–3 months of age with the following clinical picture were noted on the farm with the following clinical picture: depression, loss of appetite, serous discharge from the nose, cough and diarrhea. The number of young cattle in the farm is 180 goals, the death rate among sick young animals reached 15%.

Animals were vaccinated at a dose of 2 cm ³ . The blood serum of animals was examined for the presence of antibodies in ELISA. The selection of blood serum was carried out according to the following scheme: before vaccination, before revaccination (on day 21) and after revaccination (on day 35).

The research results are presented in table 7. According to the data of table 7 it is seen that the vaccine against cattle VD adsorbed inactivated from the strain "NADL-ARRIAH" has a high antigenic activity. The use of the vaccine allowed to reduce the mortality rate of young animals on the farm from 15% to 3%.

Example 6

Tests of antigenic activity of series No. 4 of the proposed vaccine against VD of cattle, made as described in example 2, and containing, wt.%:

Antigenic material 60.0 GOA 39.6 Saponin 0.4

Tests of the drug were carried out on the premises of ZAO Suvorovskoye in the Suzdal District of Vladimir Region. The proposed vaccine was administered intramuscularly to cows intramuscularly in the middle third of the neck: the first vaccination - 40 ÷ 45 days before calving, the second - 20 ÷ 25 days after the primary immunization.

The research results are presented in table 8. According to the data in table 8 it is seen that the vaccine against cattle VD adsorbed inactivated from the strain "NADL-ARRIAH" has a high antigenic activity.

Example 7

Tests of antigenic activity of series No. 2 of the proposed vaccine against VD of cattle, made as described in example 2, and containing, wt.%:

Antigenic material 70.0 GOA 29.6 Saponin 0.4

The antigenic activity of the vaccine against cattle VD adsorbed inactivated from the strain “NADL-ARRIAH” was studied in a series of comparative experiments on calves and deep-shelled cows in various farms in several regions of the Russian Federation.

The research results are presented in table 9. The results of observations of vaccinated animals indicate that the proposed vaccine does not cause complications and induces a fairly good resistance to the disease.

Information sources

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5. RF patent No. 1789219; A61K 39/118, 39/12; 01/23/1993

6. RF patent No. 1835659; A61K 39/15, G01N 33/556; May 27, 1996

7. Bakhtukhanov Yu.Kh., Gizitdinov NN, Study of the tension and duration of immunity in calves vaccinated with a vaccine against viral diarrhea in cattle // Prevention and control measures against infectious diseases of farm animals in Kazakhstan. - Proceedings of the Kazakh. NIVI. - T. XVIII. - Alma-Ata. - 1979. - P.81 ÷ 83.

8. RF patent No. 2362585; A61K 39/295, C12N 7/00, A61P 31/12; 07/27/2009

9. RF patent No. 2372938; A61K 39/295, A61P 31/12; November 20, 2009 (prototype).

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

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

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

Table 4 The optimal component composition (wt.%) Sorbed
inactivated VD vaccine for cattle prepared from the strain "NADL-ARRIAH" Ingredients Minimum Average Maximum Antigenic material fifty 70 80 GOA 49.6 29.6 19.6 Saponin 0.4 0.4 0.4

Table 5 Antigenic activity of the vaccine against VD of cattle adsorbed inactivated from the strain "NADL-ARRIAH" in guinea pigs Number of animals Name of material ELISA antibody titer (log ₂ ) n = 10 Guinea pig vaccinated serum 10.6 ± 0.33 n = 10 - “- 9.2 ± 0.4

Table 6 Antigenic activity of the vaccine against VD of cattle adsorbed inactivated from the strain "NADL-ARRIAH" in rabbits No. p / p Name of material ELISA antibody titer (log ₂ ) one Vaccinated rabbit serum 9.4 ± 0.23 2 - “- 10.6 ± 0.16 3 - “- 9.8 ± 0.26 four - “- 8.6 ± 0.33 average - “- 9.6 ± 0.24

Table 7 The test results on 14 ÷ 30-day-old calves of the vaccine against VD of cattle adsorbed inactivated from the strain "NADL-ARRIAH" Immune status The average titer in ELISA (log ₂ ) to the virus VD cattle Before vaccination 8.6 ± 0.92 Before revaccination 9.1 ± 0.57 After booster 10.2 ± 0.35

Table 8 The test results of the vaccine against VD of cattle adsorbed inactivated from the strain "NADL-ARRIAH" on deep-shelled cows Immune status The average titer in ELISA (log ₂ ) to the virus VD cattle Before vaccination 0 Before revaccination 9.2 ± 0.58 After booster 10.2 ± 0.41

Table 9 The level of antibodies to the VD virus of cattle in calves and deep-bred cows vaccinated with inactivated sorbed vaccine against VD of cattle from the strain "NADL-ARRIAH" No. p / p Animal blood serum Number of animals ELISA antibody titer (log ₂ ) one calves before vaccination 57 7.1 ± 0.81 2 calves after revaccination on day 35 57 9.9 ± 0.41 3 cows before vaccination 53 8.6 ± 0.67 four cows 10-15 days before calving 53 10.7 ± 0.41

Claims (13)

1. The vaccine against viral diarrhea of cattle (VD cattle), containing the active substance and target additives, characterized in that as the active substance it contains avirulent and purified antigenic material from the strain "NADL-ARRIAH" virus VD cattle, this. Flaviviridae, of the genus Pestivirus, deposited in the collection of the Federal State Institution “VGNKI” under the registration number “NADL-ARRIAH” - DEPT, in an effective amount. 2. The vaccine according to claim 1, characterized in that it contains avirulent and purified antigenic material from the strain "NADL-ARRIAH" virus VD cattle obtained in a transplantable culture of mammalian cells in an effective amount. 3. The vaccine according to claim 2, characterized in that it contains an avirulent and purified antigenic material from a strain of "NADL-ARRIAH" virus VD cattle obtained in transplantable cell culture of Taurus-2 calf kidney cells in an effective amount. 4. The vaccine according to claim 3, characterized in that it contains avirulent and purified antigenic material from the strain "NADL-ARRIAH" virus VD cattle obtained in transplantable cell culture of Taurus-2 with an infectious activity of at least 6.0 lg TCD ₅₀ / cm ³ and activity in an enzyme immunoassay (ELISA) of at least 6.6 log ₂ before inactivation, in an amount, wt.%: 50.0 ÷ 80.0. 5. The vaccine according to claim 2, characterized in that it contains an avirulent and purified antigenic material from the strain "NADL-ARRIAH" virus VD cattle obtained in the transplanted cell culture of kidney cells of saiga PS, in an effective amount. 6. The vaccine according to claim 5, characterized in that it contains avirulent and purified antigenic material from the strain "NADL-ARRIAH" virus VD cattle obtained in transplantable cell culture PS with an infectious activity of not less than 6.0 lg TCD ₅₀ / cm ³ and activity in ELISA not less than 6.6 log ₂ before inactivation, in the amount, wt.%: 50.0 ÷ 80.0. 7. The vaccine according to claim 2, characterized in that it contains an avirulent and purified antigenic material from the strain "NADL-ARRIAH" virus VD cattle obtained in a transplanted culture of kidney cells of calf RBT, in an effective amount. 8. The vaccine according to claim 7, characterized in that it contains avirulent and purified antigenic material from the strain "NADL-ARRIAH" virus VD cattle obtained in a transplanted RBT cell culture with an infectious activity of not less than 6.0 lg TCD ₅₀ / cm ³ and activity in ELISA not less than 6.6 log ₂ before inactivation, in the amount, wt.%: 50.0 ÷ 80.0. 9. The vaccine according to claim 1, characterized in that from the target additives it contains an adjuvant-sorbent aluminum hydroxide (GOA). 10. The vaccine according to claim 9, characterized in that it contains GOA in an amount, wt.%: 19.6 ÷ 49.6. 11. The vaccine according to claim 1, characterized in that from the target additives it additionally contains an adjuvant saponin. 12. The vaccine according to claim 11, characterized in that it contains saponin in an amount of 0.4 wt.%. 13. The vaccine according to any one of claims 1 to 12, characterized in that it contains avirulent and purified antigenic material from the strain "NADL-ARRIAH" virus VD cattle obtained in a transplantable culture of mammalian cells taken from the group containing Taurus-2 cell culture, PS cell culture and RBT cell culture, with infectious activity of at least 6.0 lg TCD ₅₀ / cm ³ and ELISA activity of at least 6.6 log ₂ , and target GOA and saponin supplements in the ratio, wt .%:
Antigenic material 50.0 ÷ 80.0 GOA 19.6 ÷ 49.6 Saponin 0.4