RU2395297C1 - Inactivated combined vaccine against cattle infectious rhinotracheitis, virus diarrhoea and leptospirosis - Google Patents

Abstract

FIELD: medicine. ^ SUBSTANCE: invention refers to biotechnology. The vaccine contains an active substance and target additives. The active substance is presented with mixed inactivated viral suspension of cattle infectious rhinotracheitis virus strain, SVC (State Virus Collection) No. 2333, Herpesviridae family, Alphaherpesviridae subfamily, inactivated viral suspension of cattle diarrhoea virus strain SVC No. 2336, Flaviviridae family, Pestivirus genus with activity of each strain not less than 107.0 "ö50" per 1 cm3 of the vaccine. As said active substance, the vaccine also contains mixed inactivated suspension of Leptospira interrogans bacteria cells of Pomona serogroup of strain VGNKI-6, inactivated suspension of Leptospira interrogans bacteria cells of Tarassovi serogroup of strain VGNKI-4, inactivated suspension of Leptospira interrogans bacteria cells of Grippotyphosa serogroup of strain VGNKI-1, inactivated suspension of Leptospira interrogans bacteria cells of Sejroe serogroup of Hardjo strain and inaktivirovannoj suspensions of cells of bacteria Leptospira interrogans of Sejroe serogroup of strain VGNKI-5 taken in the ratio equal to final concentration of each Leptospira strain not less than 270 million microbial cells per 1 cm3 of the vaccine. The vaccine is intended for prevention of cattle infectious diseases accompanied by reproductive disorders of various severities. ^ EFFECT: vaccine is better than common vaccines in the spectrum of specific protection and effectiveness for tolerant group of animals, duration of intense immunity, harmlessness for incalvers and newborn calves, stability of biological properties, including antigen and immunogen activity throughout 18 months (follow-up period). ^ 3 cl, 8 tbl, 6 ex

Description

The invention relates to the field of virology, biotechnology and veterinary medicine and for the creation of new vaccines that can be used to prevent infectious diseases of cattle (cattle), accompanied by impaired reproductive system functions of varying severity.

Infectious diseases causing abortion, the birth of non-viable offspring, idleness and other pathologies of the reproductive system are a serious problem of dairy and beef cattle breeding.

As a rule, these diseases are caused by several pathogens belonging to different taxonomic categories. The leading role in the pathology of reproduction belongs to the bacteria Leptospira interrogans of the serogroups Pomona, Grippotyphosa, Tarassovi, Sejroe. They are found throughout Russia and in most countries of the world. On dysfunctional farms, from 27 to 92% of the animals studied have specific antibodies to leptospira (Y. A. Malakhov, A. N. Panin, G. L. Soboleva, 2001). Leptospira-induced abortions can occur at different times after infection and at any stage of pregnancy. Sometimes abortion is the only clinical sign of infection (Ellis, 1985, Thiermann, 1985).

In addition to leptospira in the development of infectious abortion, a certain etiological significance also belongs to the viruses of infectious rhinotracheitis (IRT), viral diarrhea (VD), rota- (RV) and coronaviruses (KB). Diseases caused by mixed infection are clinically more severe, longer, and along with impaired reproductive function are characterized by damage to the respiratory and gastrointestinal tracts.

For the prevention of infectious abortion in cattle, the multivalent vaccine VGNKI against animal leptospirosis, made in two versions and containing a formalin-inactivated suspension of serogroup Leptospira interrogans strains, Pomona PSR-1, Tarassovi VGNKI-4, IcterohaemorrHagiGanIe, was widely used. 3 (1st option) or Pomona PSR-1, Tarassovi VGNKI-4, Grippotyphosa VGNKI-1, Sejroe "Hardjo" (2nd option), with a total concentration of 0.5-5.0 billion microbial cells in each variant in 1 cm ³ , adjuvant - alumina hydrate, and stabilizer (Patent RU No. 2030915, А61К 3 9/02, published on March 20, 1995).

The disadvantage of the multivalent vaccine VGNKI is that it does not provide immunological protection for animals against infectious abortions of viral etiology due to the absence of infectious rhinotracheitis virus antigens and viral diarrhea in the vaccine.

Known associated vaccine against leptospirosis and campylobacteriosis of cattle, designed to prevent diseases that cause impaired reproduction. The vaccine contains leptospira antigens of serological groups Pomona, Tarassovi, Grippotyphosa, Sejroe and campylobacter antigens from strains VGNKI N 43/3 and VGNKI N 169/17, inactivator and adjuvant (Patent RU No. 2021818, A61K 39/116, publ. .

A disadvantage of the known associated vaccine is the limited spectrum of action, since it is directed against a narrow group of pathogens and does not provide immunological protection of animals against infectious abortions of viral etiology.

Also known is a vaccine composition against infectious diseases of cattle, accompanied by damage to the reproductive organs and respiratory tract, containing attenuated bovine herpes virus (infectious rhinotracheitis) and at least one antigen selected from the group consisting of: viral diarrhea virus; attenuated parainfluenza-3 virus; attenuated respiratory syncytial virus; bacteria Leptospira canicola, Leptospira grippotyphosa, Leptospira borgpetersenii hardjo-prajitno, Leptospira icterohaemorrhagiae, Leptospira interrogans pomona, Leptospira borgpetersenii hardjo-bovis, Leptospira bacterius bacillus ul. .

However, this vaccine composition is characterized by low antiepizootic efficacy due to the fact that the strains used for its manufacture possess antigenic and immunobiological differences compared with similar characteristics of epizootic pathogens circulating in Russia and the CIS countries.

In addition, the use of live vaccines containing uninactivated antigens of the viruses of infectious rhinotracheitis and viral diarrhea is categorically contraindicated for pregnant cows and newborn calves because of the possibility of homologous recombination of vaccine and field strains of the virus, leading to exacerbation of infections, abortions and even death of individual animals.

The closest analogue is a vaccine against infectious diseases causing abortion in cattle, containing attenuated bovine herpes virus (infectious rhinotracheitis), attenuated parainfluenza-3 virus, attenuated respiratory syncytial virus, viral diarrhea virus; and at least one antigen selected from the group consisting of Leptospira canicola, Leptospira grippotyphosa, Leptospira borgpetersenii hardio-prajitno, Leptospira icterohaemorrhagiae, and Leptospira interrogans pomona, Leptospira borgpetersenii hardptobosporis bis and a pharmaceutically acceptable carrier (International application WO 2004017990, A61K 39/02, published 04.03.2004).

However, this vaccine does not contain antigens against pathogens circulating in Russia and the CIS countries, and as a result is characterized by low antiepizootic efficacy.

The objective of the invention is to develop a safe combined (multivalent) vaccine containing antigenically and immunogenically balanced viral and bacterial antigens, which will create intense immunity against the most significant pathogens associated with damage to the reproductive system, and thus ensure the safety of young animals and adult productivity animals.

Another objective is to obtain new production strains of the IRT and VD viruses with a wide antigenic spectrum, high antigenic and immunogenic activity not only in their native form, but also after inactivation, stably preserving their properties, and suitable for creating inactivated combined vaccines of different valencies.

The technical result of the invention is to expand the arsenal of combined vaccines against infectious diseases, accompanied by damage to the reproductive system, as well as to increase the stability, antigenic and immunogenic activity of the combined vaccine and create intense and lasting immunity in vaccinated animals and born calves against infectious rhinotracheitis, viral diarrhea and leptospirosis cattle.

The technical result of the invention is achieved by using new vaccine strains of infectious rhinotracheitis viruses and cattle viral diarrhea with a wide antigenic spectrum in the vaccine, as well as by creating a synergistic composition of a multivalent vaccine when combining new production virus strains with known leptospira vaccine strains.

The invention consists in the following.

The proposed inactivated combination vaccine against infectious rhinotracheitis, viral diarrhea and leptospirosis in cattle, containing the active substance and target additives, is characterized in that as the active substance it contains a mixture of inactivated suspension of the strain of infectious rhinotracheitis of cattle STV No. 2333, family Herpesviridae, subfamily Alphaherpesviridae, inactivated suspension of the strain of bovine diarrhea virus of bovine bacilli No. 2336, family Flaviviridae, genus Pestivirus, inactivated suspensions of bacterial cells of Leptospira interrogans serogroup Pomona strain VGNKI-6, an inactivated suspension of bacterial cells of bacteria Leptospira interrogans serogroup Tarassovi strain VGNKI-4, an inactivated suspension of bacterial cells of Leptospira interrogans serogroup Grippotyphosa strain of serogroup Septogens of the bacterium leptospherosis of the bacterium of the bacterium Lepto bacteria and an inactivated suspension of bacterial cells of the Leptospira interrogans serogroup Sejroe strain VGNKI-5, in an effective amount.

The term “effective amount” means an amount of an active substance which, when administered to cows and / or calves, provides the induction of a specific immune response of a sufficient level in animals, followed by a warning against the disease and the symptoms of the disease to be prevented.

The bovine infectious rhinotracheitis virus strain of bovine bovine vaccine No. 2333, the family Herpesviridae, subfamily Alphaherpesviridae, and the bovine diarrhea virus strain of bovine bovine vaccine No. 2336, family Flaviviridae, genus Pestivirus, are new production strains of the vaccines and are used for the first time .

The term "production strain" means a vaccine strain, studied by its biological properties, stable during storage and replanting, harmless after inactivation or attenuation for the animal, well adapted to the cultivation system, with high antigenic and immunogenic activity, both for a sensitive laboratory model and for naturally susceptible animals, and also characterized by a wide antigenic spectrum that dominates epizootic strains of the pathogen.

The production strain of the infectious rhinotracheitis virus of cattle, STB No. 2333, was deposited in the State Virus Collection of the Institute of Virology of the Russian Academy of Medical Sciences, has the registration number of the STB participant No. 2333 and is characterized by the following features and properties.

Source of Origin:

The source of the production of a new production strain of the cattle infectious rhinotracheitis virus is the B-25 virus (author's name), isolated in 1991 from a nasal flush of a calf with respiratory syndrome, which passed 35 consecutive passages in cattle transplant cultures.

Morphological properties:

GKV strain No. 2333 belongs to the family Herpcsviridae, subfamily Alphaherpesviridae. An electron microscopic examination using the method of negative contrasting of the virus-containing culture material revealed virions characteristic of the cattle infectious rhinotracheitis virus, 150-200 nm in size and consisting of a capsid of cubic symmetry and an outer shell covering it. Capsid has 162 prismatic hollow capsomeres. A nucleocapsid is formed inside the nucleus, where type A inclusions are formed.

Antigenic properties:

GKV strain No. 2333 shows high antigenic and immunogenic activity both in the native form and after inactivation, has a wide antigenic spectrum and dominates in relation to field strains.

The introduction of guinea pigs strain GKV No. 2333 of the IRT virus leads to the formation of a humoral immune response to various strains and field isolates of the IRT virus (table 1).

Table 1 Cross antigenic activity of strain GKV No. 2333 virus IRT RTI virus strain / isolate for pH The titer of virus-neutralizing antibodies in the blood serum of guinea pigs immunized with strain ГКВ №2333 of the virus ИРТ *, М ± m ГКВ №2333 142.1 ± 24.2 B-25 84.4 ± 13.8 Isolate No. 1702 76.1 ± 23.9 Isolate No. 2105 87.3 ± 12.5 Isolate No. 1320 111.4 ± 26.5 * hereinafter in the tables the reverse values of antibody titer are given

Biotechnological characteristics:

GKV strain No. 2333 proliferates well in primary and transplanted cell cultures (kidneys, testicles) obtained from tissues and organs of cattle. In these cell cultures, the virus accumulates in a titer of 10 ^7.5 -10 ^8.5 TCD ₅₀ / cm ³ .

Recommended storage method:

In a lyophilized state at a temperature not exceeding minus 60 ° С.

Frequency of refreshment:

Once every three years in a transplanted culture of MDVC calf kidney cells (hereinafter referred to as MDVC cell culture).

Additional information about the strain:

GKV strain No. 2333 is not contaminated by extraneous microflora. Tested with reference serum (GB).

Reactogenicity - strain GKV No. 2333 does not have reactogenic properties.

Stability - preserves the initial biological (antigenic) properties during successive passages in the MDVC cell culture for 10 passages (observation period).

Pathogenicity - expressed.

Contagiousness - expressed.

Oncogenicity is absent.

The bovine bovine diarrhea virus production strain No. 2,336 was deposited with the State Virus Collection Institute of the Institute of Virology of the Russian Academy of Medical Sciences, has the registration number of No. 2,336 bills and is characterized by the following features and properties.

Source of Origin:

The source of the new production strain of STB diarrhea virus No. 2336 is the ST-89 virus strain (author's name), isolated in 1989 from the intestines of calves with signs of diarrhea in the cell culture of the kidney of a newborn calf, which passed 82 consecutive passages in a transplanted cell culture of MDVC .

Morphological properties:

GKV strain No. 2336 belongs to the family Flaviviridae, genus Pestivirus. Electron microscopic examination using the method of negative contrasting of virus-containing culture material revealed virions characteristic of the cattle diarrhea virus, 40-60 nm in size and consisting of icosahedral symmetric nucleocapsid and external lipoprotein membrane.

Antigenic properties:

GKV strain No. 2336 shows high antigenic and immunogenic activity both natively and after inactivation, possesses a wide antigenic spectrum and dominates against field strains.

The introduction of Guinea pigs strain No. 2,336 of the cattle diarrhea virus leads to the formation of a humoral immune response to various strains and field isolates of the VD virus (Table 2).

Table 2 Cross antigenic activity of strain GKV No. 2336 of the IRT virus VD strain / isolate for pH The titer of virus-neutralizing antibodies in the blood serum of guinea pigs immunized with the strain of STB No. 2336 of the VD virus, M ± m ГКВ №2336 374.8 ± 31.6 ST-89 107.6 ± 24.5 Isolate No. 865 168.9 ± 21.3 Isolate No. 1204 337.7 ± 26.8 Isolate No. 663 174.8 ± 22.4

It has a close antigenic relationship with the viruses of classical swine fever and border disease of sheep.

Biotechnological characteristics:

GKV strain No. 2336 multiplies well in the primary culture of calf kidney cells (cow embryos) and in the MDVK cell culture. In these cell cultures, the virus accumulates in a titer of 10 ^7.5 -10 ^8.5 TCD ₅₀ / cm ³ .

Recommended storage method:

In a lyophilized state at a temperature not exceeding minus 60 ° С.

Frequency of refreshment:

Once every three years in a culture of MDVC cells.

Additional information about the strain:

GKV strain No. 2336 is not contaminated by extraneous microflora. It is tested with reference serum (GB) and in the PCR test system.

Reactogenicity - the strain of ST-Bills No. 2336 does not have reactogenic properties.

Stability - preserves the initial biological (antigenic) properties during successive passages in the MDVC cell culture for 10 passages (observation period).

Pathogenicity - expressed.

Contagiousness - expressed.

Oncogenicity is absent.

As part of a combined vaccine against infectious rhinotracheitis, viral diarrhea and cattle leptospirosis, inactivated viral suspensions of the STB strains No. 2333 and STB No. 2336 are contained with an infectious activity titer of each virus of at least 10 ^7.5 TCD ₅₀ in 1 cm ^{3 of the} vaccine.

Inactivation of viral suspensions is carried out in any known manner, for example by adding formalin or ethyleneimine dimer (DEI).

In the composition of the proposed vaccine, viral suspensions of the strains can be used in any combination by volume, for example, viral suspensions are contained in a ratio of 1: 1 by volume with the titer of infectious activity of strains of GKV No. 2333 and GKV No. 2336 - 10 ^8.0-8.5 TCD ₅₀ / cm ³ . In other embodiments of the preparation of the vaccine, viral suspensions are contained in a ratio of 1: 2 if the infectious activity of the STB strain No. 2333 is 10 ^8.5-9.0 TCD ₅₀ / cm ³ , and the STB strain No. 2333 is 10 ^7.5-8.0 TCD ₅₀ / cm ³ ; or in a ratio of 2: 1 in volume, if the infectious activity of the STB strain No. 2333 is 10 ^7.5-8.0 TCD ₅₀ / cm ³ , and the STB strain No.2336 is 10 ^8.0-8.5 TCD ₅₀ / cm ³ . It should be noted that an increase or decrease in the volume of one of the components does not significantly affect the antigenic activity of the finished vaccine.

The strains of Leptospira interrogans used for the manufacture of the leptospirosis component of the combination vaccine are known and used in the manufacture of the multivalent vaccine VGNKI against animal leptospirosis. However, the authors found that when combining the known strains of leptospira with new production strains of viruses in the combination according to the invention, the formation of a synergistic composition is observed, which is manifested in an increase in tension and duration of immunity to the viral and leptospirosis components of the vaccine.

The composition of the proposed vaccine inactivated cell suspensions of bacteria Leptospira interrogans serogroup Pomona (strain VGNKI-6), serogroup Tarassovi (strain VGNKI-4), serogroup Grippotyphosa (strain VGNKI-1) and serogroup Sejroe (strains "Hardjo" and VGNKI-5) contained in equal proportions with a final concentration of at least 270 million microbial cells in 1 cm ^{3 of the} vaccine.

Inactivation of leptospira cell suspensions is carried out by any known method, for example, by adding formalin containing at least 37% formaldehyde.

According to the invention, the combination vaccine also contains one or more veterinary suitable target additives.

The term “target additive” means any pharmaceutical components acceptable in veterinary medicine that are traditionally used to ensure the preservation of antigens during processing, preparation of the final prescription form or subsequent storage and the like.

As a targeted additive, the combination vaccine may contain a solvent, an adjuvant, a solubilizing agent, a diluent, a preservative, a stabilizer, an antibacterial and antifungal agent, an isotonic agent, an absorption delaying agent, and the like. Diluents may include distilled water, saline, saline, dextrose, ethanol, glycerol and the like. Isotonic agents may include, among others, sodium chloride, dextrose, mannitol, sorbitol, and lactose. Stabilizers include albumin, sucrose, gelatose, skim milk, etc.

Adjuvants include substances that non-specifically enhance the immune response to antigens, and may be of mineral origin — alumina hydrate, aluminum phosphate, potassium alum; plant origin - saponin (Quil-A); adjuvants based on mineral and non-mineral oils; synthetic adjuvant - muramyl dipeptide, etc.

As a target additive, the proposed vaccine mainly contains an adjuvant, for example, alumina hydrate or a mixture of alumina hydrate with saponin.

If necessary, as a target additive, the proposed vaccine may contain a preservative sodium thiolate in an amount of 1: 10000.

Although a person skilled in the art can easily determine the amount and concentration of adjuvants and additives that can be used in the context of the present invention, the present invention relates to compositions comprising preferably from 20 to 30 vol.% Adjuvant.

In a preferred embodiment, the vaccine contains 6% alumina hydrate gel in an amount of 20 vol.% And saponin in an amount of 0.05 vol.%, Which corresponds to 12 g of alumina hydrate and 500 mg of saponin in 1 liter of vaccine.

The following formulation examples are illustrative only and do not limit the scope of the present invention.

The inactivated combined vaccine against infectious rhinotracheitis, viral diarrhea, and cattle leptospirosis is a light red liquid; upon storage, a loose grayish precipitate forms, which easily breaks up into a homogeneous suspension with shaking.

The vaccine is stable and retains its original activity at a temperature of 2-8 ° C for at least 18 months (observation period).

In order to prevent abortion, the resulting vaccine is administered to adult animals twice with an interval of 14-21 days, a second time no later than 2 weeks before insemination. Pregnant animals are vaccinated twice: the first time - 50-60 days before calving, the second time - 14-21 days after the first immunization (no later than 30 days before calving). Calves aged 40-45 days are vaccinated twice with an interval of 20-25 days. Revaccination is carried out once in the same dose, every 6 months. The vaccine is administered intramuscularly to adult animals at a dose of 3 cm ³ , calves at a dose of 2 cm ³ .

The vaccine was tested on cattle in dysfunctional farms. This testing confirms the harmlessness of the drug and its antiepizootic efficacy, manifested in enhancing the immunizing activity of the components of the inactivated vaccine. Vaccination increases immunity in mothers and protects offspring during embryonic development and in the postnatal period. Moreover, the induced immune response was characterized by high specificity with respect to different subtypes and main antigenic variants of epizootic strains of pathogens circulating on farms.

The protective level of antibodies in animals vaccinated with this vaccine was maintained for at least 8 months in adult animals and 6 months in young animals under the age of 1 year, colostral immunity - up to 1.5 months.

The invention is illustrated, but not limited to the following examples.

Example 1. Obtaining an inactivated combination vaccine against infectious rhinotracheitis, viral diarrhea and leptospirosis of cattle.

The vaccine is prepared from new production strains of the infectious rhinotracheitis virus GKV No. 2333, diarrhea virus GKV No. 2336 (viral component) and the known vaccine strains of Leptospira interrogans leptospira serogroup Pomona (VGNKI-6 strain), serogroup Tarassovi (serogroup Gphi-4, strain VGNfzy-4) strain VGNKI-1) and serogroup Sejroe (strains "Hardjo" and VGNKI-5) (leptospirosis component).

The technological process consists of the following main stages: the stage of obtaining the viral component, the stage of obtaining the leptospirosis component, combining the obtained viral and leptospirosis components, adding an adjuvant, mixing all components of the vaccine with subsequent packaging of the finished product.

A. Stage of obtaining the viral component of the combination vaccine.

The manufacture of the viral component of the combination vaccine includes separate steps for the preparation of viral suspensions from seeds of the strains of the infectious rhinotracheitis virus GKV No. 2333 and the diarrhea virus GKV No. 2336; determination of the infectious activity of each viral suspension, inactivation of virus-containing culture fluids of each strain and their combination.

The step for producing viral suspensions according to the invention is based on a seed lot system, in which successive series of products are obtained from the main seed (master seed lot) for a certain number of transfers (passages). In the current production, the working seed lot is prepared from the main seed. The finished product is made from working seed, and the number of transfers from the main seed should not exceed the value established during clinical trials of vaccines, based on safety and efficacy requirements.

The process of obtaining a seed system of each strain of the virus includes several stages:

- preparation of cell culture;

- carrying out passages of the virus;

- obtaining a viral suspension;

- intermediate control of a strain of the virus;

- lyophilization, freezing or inactivation of the virus strain;

- control of the strain of the virus.

The strains of infectious rhinotracheitis viruses ГКВ №2333 and viral diarrhea ГКВ №2336 are grown separately in the transplanted culture of MDVK calf kidney cells.

The accumulation of viruses in cell culture is assessed by the cytopathic effect. The virus titer is calculated by the method of Reed and Mench and expressed in lg TCD ₅₀ / cm ³ .

When the virus titer reaches values of 10 ^7.5 -10 ^9.0 TCD ₅₀ / cm ³ , the resulting virus-containing material of each strain is purified from ballast impurities by centrifugation or any other known method.

Further, virus suspensions are inactivated with a formalin solution containing at least 37% formaldehyde. The completeness of inactivation is assessed by the absence of virus propagation in a sensitive cell culture for four passages.

Inactivated virus suspensions are combined so that in each series the titer of the infectious activity of each of the strains is at least 10 ^7.5 TCD ₅₀ / cm ³ , for example, the combined viral suspensions are prepared in the following proportions:

Stage A (1) - mix the viral suspension of the STB strain No.2333 and the STB strain No.2336 in a 1: 1 ratio by volume if the infectious activity titer of each strain is 10 ^8.0-8.5 TCD ₅₀ / cm ³ ;

Stage A (2) - mix the viral suspension of the STB strain No. 2333 and the STB strain No. 2336 in a ratio of 1: 2 by volume, if the infectious activity of the STB strain No. 2333 is 10 ^8.5-9.0 TCD ₅₀ / cm ³ , and the strain STB No. 2333 - 10 ^7.5-8.0 TCD ₅₀ / cm ³ ;

Stage A (3) - mix the viral suspension of the STB strain No.2333 and the STB strain No.2336 in a ratio of 2: 1 in volume, if the infectious activity of the STB strain No.2333 is 10 ^7.5-8.0 TCD ₅₀ / cm ³ , and the strain ГКВ №2336 - 10 ^8.0-8.5 TCD ₅₀ / cm ³ .

Next, the combined viral suspensions are thoroughly mixed.

B. The stage of obtaining the leptospirosis component of the combination vaccine.

Cultures of Leptospira interrogans leptospira serogroup Pomona (strain VGNKI-6), serogroup Tarassovi (strain VGNKI-4), serogroup Grippotyphosa (strain VGNKI-1) and serogroup Sejroe (strains "Hardjo" and VGNKI-5) are grown separately in liquid nutrient medium within 7-10 days before the accumulation of each strain of leptospira, at least 70 million microbial cells in 1 cm ³ .

Next, the resulting cultures are combined in the ratios 1: 1: 1: 0.5: 0.5, formalin is added to a concentration of 0.15%. The resulting mixture of inactivated cell suspensions is concentrated by ultrafiltration to obtain a backmass containing 2 billion microbial cells in 1 cm ³ or more.

B. Stage of preparation of the finished drug combination vaccine.

The combination vaccine according to the invention is obtained by combining the viral and leptospirosis components of the vaccine and then adding targeted additives.

5 experimental series (options) of the vaccine are made.

Option 1.

To the combined inactivated suspension of viruses prepared in stage A (1), add 6% gel of aluminum hydroxide (GOA) in an amount of 20% of the total volume, then add saponin at a concentration of 500 mg / l and a preservative sodium merthiolate at a concentration of 1: 10000, mix thoroughly.

Sodium merthiolate at a concentration of 1: 10000 as a preservative is added to an inactivated bacterial cell suspension of the mixture of leptospira strains prepared in stage B, then mixed with 6% GOA (20% of the total volume), mixed thoroughly.

The viral and leptospirosis components of the drug are mixed so that the concentration of viral antigens in the finished vaccine is at least 7.5 lg TCD ₅₀ , and leptospira is at least 270 million cubic meters. in 1 cm ³ .

Option 2

To the combined inactivated suspension of viruses prepared in stage A (2), add 6% aluminum hydroxide gel (GOA) in an amount of 20% of the total volume, then add saponin at a concentration of 500 mg / l and a preservative sodium merthiolate at a concentration of 1: 10000, mix thoroughly.

To inactivated bacterial cell suspension of the mixture of leptospira strains prepared in stage B, add 6% GOA (20% of the total volume) and mix thoroughly.

The viral and leptospirosis components of the drug are mixed so that the concentration of viral antigens in the finished vaccine is at least 7.5 lg TCD ₅₀ , and leptospira is at least 270 million cubic meters. in 1 cm ³ .

Option 3

To the combined inactivated virus suspension prepared in stage A (3), add 6% gel of aluminum hydroxide (GOA) in an amount of 20% of the total volume, add saponin at a concentration of 500 mg / l and a preservative sodium merthiolate at a concentration of 1: 10000 mix thoroughly.

To inactivated bacterial cell suspension of the mixture of leptospira strains prepared in stage B, add 6% GOA (20% of the total volume) and mix thoroughly.

The viral and leptospirosis components of the drug are mixed so that the concentration of viral antigens in the finished vaccine is at least 7.5 lg TCD ₅₀ , and leptospira is at least 270 million cubic meters. in 1 cm ³ .

Option 4

To the combined inactivated virus suspension prepared in stage A (1), add 6% aluminum hydroxide gel (GOA) in an amount of 20% of the total volume, then add saponin at a concentration of 500 mg / l, mix thoroughly.

To an inactivated bacterial cell suspension of a mixture of leptospira strains prepared in stage B and having a concentration above 2 billion cubic meters, 6% GOA (20% of the total volume) is added and mixed thoroughly.

The viral and leptospirosis components of the drug are mixed so that the concentration of viral antigens in the finished vaccine is at least 7.5 lg TCD ₅₀ , and leptospira is at least 270 million cubic meters. in 1 cm ³ .

Option 5

To the combined inactivated virus suspension prepared in stage A (1), add 6% aluminum hydroxide gel (GOA) in an amount of 30% of the total volume.

To an inactivated bacterial cell suspension of a mixture of leptospira strains prepared in stage B and having a concentration above 2 billion cubic meters, 6% GOA (30% of the total volume) is added and mixed thoroughly.

The viral and leptospirosis components of the drug are mixed so that the concentration of viral antigens in the finished vaccine is at least 7.5 lg TCD ₅₀ , and leptospira is at least 270 million cubic meters. in 1 cm ³ .

The obtained variants of the inactivated combined vaccine against infectious rhinotracheitis, viral diarrhea and leptospirosis in cattle have a component composition shown in Table 3.

Table 3 The composition of the combined vaccine against infectious rhinotracheitis, viral diarrhea and leptospirosis in cattle Ingredient Titer, lg TCD ₅₀ / cm ³ The content of ingredients, vol.% Option 1 Option 2 Option 3 Option 4 Option 5 IRT virus 7.5-8.0 - - 40 - - 8.0-8.5 thirty - - 35 thirty 8.5-9.0 - twenty - - - VD virus 7.5-8.0 - 40 - - - 8.0-8.5 thirty - twenty 35 thirty Leptospira strain cell suspension - 19.94 19.94 19.94 9.95 10.0 GOA (6% gel) - twenty twenty twenty twenty thirty Saponin - 0.05 0.05 0.05 0.05 - Mertiolate Sodium - 0.01 0.01 0.01 - -

Example 2. Evaluation of the antigenic activity of a combination vaccine in experimental models.

To determine the antigenic activity of the combination vaccine according to the invention, laboratory animals are used as an experimental model, while guinea pigs weighing 350-400 g and leptospirosis component rabbits weighing 3-3.5 kg are used to evaluate the antigenic activity of the viral components of the vaccine.

Groups of 10 animals are formed in each: 3 experimental groups and 1 control (guinea pigs), 3 experimental groups and 1 control (rabbits).

Guinea pigs of the experimental groups are immunized with the test vaccine at a dose of 0.25 cm ³ ; 0.5 cm ³ and 1.0 cm ^3, respectively, for rabbits, in a dose containing at least 15 million leptospira of each serogroup. In this case, the vaccine is administered intramuscularly to guinea pigs, and intravenously to rabbits. Animal control groups are not immunized.

On the 21st day after the injection of the vaccine, blood was taken from all animals and the serum was separated, which was then examined for the presence of specific antibodies.

The antigenic activity of the viral component of the vaccine is evaluated by the level of virus-specific antibodies to the viruses of IRT and VD. The antibody titer is determined in the neutralization reaction (PH), which is carried out in 96-well plates according to the standard method by double dilutions of the test serum against a working dose of each virus of 100 TCD ₅₀ / well.

The virus neutralizing titer of the test serum is expressed as its limiting dilution, inhibiting the development of the virus's CPD in 50% of infected cell cultures.

The antigenic activity of the leptospirosis component of the vaccine is evaluated in the microagglutination reaction (PMA) by the titer of antibodies in the blood serum of vaccinated rabbits.

The results of the study are presented in table 4, which indicate that the combined vaccine against IRT, VD and cattle leptospirosis is superior to existing analogues in antigenic activity. So, the proposed vaccine already 21 days after immunization provides the production of specific antibodies to all components of the drug in high titer: to the IRT virus - 1: 79.8, to the VD virus - 1: 114.1, to leptospira - from 1: 1533 to 1: 1744, the preventive activity of serum of rabbits was 100%. The experiments confirmed that the control of the vaccine for the leptospirosis component should be carried out on rabbits at a dose of at least 15 million microbial cells of the leptospira of each serogroup.

The animals of the control group throughout the experiment were seronegative to the viruses IRT, VD and leptospira.

Table 4 Antigenic activity of a combined vaccine against infectious rhinotracheitis, viral diarrhea and lettospirosis in cattle in experimental models Live group Guinea pig serum antibody titer Batch number Leptospira rabbit serum antibody titer Preventive activity of serum of rabbits to leptospira,% The dose of the drug, cm ³ The titer of neutralizing antibodies, M ± m IRT VD I 0.25 26.1 ± 5.4 70.2 ± 9.5 one 1744 ± 256 fifty II 0.5 79.8 ± 6.2 114.1 ± 12.6 2 1680 ± 194 one hundred III 1,0 63.1 ± 5.2 91.5 ± 10.3 3 1533 ± 205 80 the control - 0 0 0 0 0

Example 3. Evaluation of the stability of the combination vaccine during storage.

The stability test of the combination vaccine obtained in example 1 is carried out on experimental animals - guinea pigs and rabbits. Moreover, to assess the stability of the antigenic activity of the viral components of the vaccine, guinea pigs and the leptospirosis component are used in rabbits.

For this, the vaccine is administered after 6, 12 and 18 months. storage at a temperature of 4 ° C once: to guinea pigs at a dose of 0.5 cm ³ , to rabbits at a dose of at least 15 million microbial cells, leptospira of each serogroup. Twenty-one days after vaccination, the blood serum of the animals was taken for examination.

The stability of the viral component of the vaccine is assessed in pH, leptospirosis in the PMA by the absence of a decrease in antibody titer below the established level (1:16 and 1: 400, respectively) when the vaccine is stored at + 4 ° C for 18 months.

The test results are presented in table.5.

Table 5 Change in antigenic activity of the combined vaccine during storage Storage time Serum antibody titer of vaccinated laboratory animals When made. 6 months 12 months 18 months IRT 79.8 ± 12.3 80.0 ± 10.2 76.5 ± 12.5 65.8 ± 8.9 VD 114.1 ± 10.8 108.2 ± 22.5 110.2 ± 16.9 95.8 ± 12.4 Leptospira 1680.8 ± 152.3 1450.2 ± 186.3 1428.5 ± 253.1 859.6 ± 106.3

Example 4. Production test of experimental series of combination vaccines in experiments on cows.

The test is carried out on cows and heifers of a random age in farms with a high level of abortion, unsuccessful for infectious rhinotracheitis, viral diarrhea and leptospirosis in cattle.

In this case, animals are divided into 2 groups: experimental and control, 20 goals in each group.

For the animals of the experimental group, the combined vaccine obtained according to Example 1 is administered intramuscularly at a dose of 3.0 cm ³ twice with an interval of 14-21 days, a second time no later than 2 weeks before insemination.

In the control group, animals are not vaccinated.

To determine the tension and duration of immunity in all animals, blood is taken before vaccination and after 1, 2, 4, 6, 8 and 12 months after vaccination.

Vaccination efficacy is evaluated by the level of virus-neutralizing antibodies in the blood serum of cows and by leptospira by the preventive activity of the blood serum of vaccinated animals for golden hamsters infected with 10 LD _{50 of the} virulent leptospira strain. The test results are presented in table.6.

Table 6 Evaluation of the activity of the vaccine against infectious rhinotracheitis, viral diarrhea and leptospirosis in cattle in an experiment on cows Research Dates Antigenic activity of a vaccine against viral antigens Leptospira vaccine immunogenic activity IRT VD Preventive activity of blood serum of vaccinated animals for golden hamsters,% Experienced group Before vaccination 32.2 ± 8.6 45.2 ± 8.7 0 1 month n / vacc. 315.5 ± 56.0 345.5 ± 35.4 70 2 months n / vacc. 286.3 ± 21.3 326.4 ± 26.8 one hundred 4 months n / vacc. 181.0 ± 16.3 252.6 ± 13.8 90 6 months n / vacc. 152.2 ± 12.8 189.4 ± 11.8 one hundred 8 months n / vacc. 91.5 ± 10.1 124.4 ± 10.7 80 12 months n / vacc. 64.9 ± 9.6 78.9 ± 9.6 70 Control group - 36.8 ± 8.5 52.6 ± 6.9 10

The results of the study show that immunization of cows with the combination vaccine according to the invention causes the formation of a pronounced immune response to all antigens that make up the vaccine for at least 8 months.

Example 5. Production test of experimental series of combined vaccines in experiments on calves.

To determine the prophylactic efficacy of the combination vaccine obtained in Example 1, 60 calves aged 40-45 days born to unvaccinated mothers were selected.

3 groups of 20 goals are formed in each: 2 experimental and control.

The calves of the experimental groups studied combination vaccine is administered intramuscularly at a dose of 1.0 and 2.0 cm ³ .

Revaccination is carried out 20-25 days after the first immunization in the same doses.

The calves of the control group are not given a vaccine.

Before each administration of the drug and 21 days after 2 vaccinations, blood was taken from all animals and the level of virus-specific antibodies in the blood serum in the PH was determined, as well as the preventive activity of the blood serum of vaccinated calves for golden hamsters infected with 10 LD _{50 of the} virulent leptospira strain.

The test results are presented in table.7.

Table 7 The level of specific antibodies in the blood serum of calves immunized with the combination vaccine of the invention Group of animals The dose of the vaccine, cm ³ The titer of neutralizing antibodies, M ± m To the virus Before vaccination 21 days after 1 vaccination 21 days after 2 vaccinations Experienced group I 1,0 IRT 7.4 ± 3.2 28.8 ± 6.2 200.9 ± 26.2 VD 16.6 ± 3.6 48.5 ± 10.2 187.4 ± 36.6 Leptospira * thirty fifty 70 II 2.0 IRT 8.6 ± 2.4 30.9 ± 8.6 394.0 ± 28.9 VD 14.4 ± 3.5 59.7 ± 6.7 274.4 ± 12.7 Leptospira * thirty 70 90 Control group - IRT 10.2 ± 2.5 12.3 ± 2.9 7.5 ± 2.1 VD 15.6 ± 3.6 14.4 ± 4.1 20.2 ± 8.3 Leptospira * twenty 10 10 * Preventive activity of serum of vaccinated calves for golden hamsters infected with 10 LD _{50 is} determined for leptospira

The data in table 7 indicate that immunization of calves of both experimental groups with the vaccine according to the invention leads to the formation of a pronounced immune response and a statistically significant increase in antibodies and preventive activity of the blood serum of vaccinated animals (P <0.01).

The maximum antibody content for all viruses is recorded on day 21 after the second vaccination of each of the two test doses of the vaccine. The highest titer of neutralizing antibodies is detected in calves with the introduction of a vaccine in a dose of 2 cm ³ . Maximum preventive activity is also expressed in calves of the same experimental group.

Example 6. Evaluation of colostral immunity in newborn calves.

In the experiments, 7-14 day old calves born to pregnant cows immunized with the combination vaccine according to the invention are used, calves of a similar age obtained from unvaccinated cows are used as controls.

All experimental and control calves receive colostrum in a timely manner from their mothers during the first two hours after birth and are kept under the same conditions.

Assessment of colostral immunity is carried out by determining the titer of specific antibodies in the blood serum of all experimental and control animals and to leptospira by the preventive activity of blood serum of calves for golden hamsters infected with 10 LD ₅₀ virulent leptospira strain.

The test results are presented in table.8.

Table 8 Evaluation of the colostral immunity of a vaccine against infectious rhinotracheitis, viral diarrhea, and cattle leptospirosis in a calf experiment Age of newborn calves, weeks Antigenic activity of a vaccine against viral antigens Leptospira vaccine immunogenic activity IRT VD Preventive activity of blood serum of vaccinated animals for golden hamsters,% Experienced group one 222.2 ± 12.6 335.9 ± 26.8 one hundred 2 198.6 ± 10.9 228.6 ± 12.7 80 four 100.8 ± 8.9 112.4 ± 10.6 fifty 6 56.6 ± 5.9 45.2 ± 10.3 40 Control group one 38.9 ± 8.6 64.0 ± 10.8 0 2 12.2 ± 2.6 10.7 ± 5.7 0 four 0 0 0 6 0 0 0

As a result of the study, it was found that vaccination of pregnant cows with the studied vaccine provides the creation of colostral immunity in calves obtained from vaccinated animals and received colostrum in time from their mothers. The formation of colostral immunity is confirmed by high titers of specific antibodies to viral components and preventive activity against leptospira, which persisted throughout the observation period.

Thus, the experiments show that the inactivated combination vaccine against infectious rhinotracheitis, viral diarrhea and leptospirosis in cattle in accordance with the invention has a decisive advantage over other known vaccines in terms of the specific protection and efficacy for a susceptible group of animals, duration of intense immunity, harmlessness for pregnant animals and newborn calves, according to the stability of the preservation of biological properties, including antigenic and immunogenic activity for 18 months (observation period). The vaccine is environmentally friendly, does not cause dissemination of pathogenic agents into the external environment.

Claims (3)

1. Inactivated combination vaccine against infectious rhinotracheitis, viral diarrhea and leptospirosis in cattle, containing the active substance and target additives, characterized in that as the active substance it contains a mixture of inactivated viral suspension of the strain of infectious rhinotracheitis of the cattle GKV No. 2333, family Herpesviridae, a subfamily of Alphaherpesviridae, an inactivated viral suspension of a strain of cattle diarrhea virus, GBV No. 2336, family Flaviviridae, genus Pestivirus, with actively Tew each of the strains at least 10 ^7,0 TCD ₅₀ per 1 cm ³ of a vaccine, inactivated bacterial cell suspension Leptospira interrogans serogroup Pomona strain VGNKI 6-inactivated suspension of bacterial cells from serogroup Leptospira interrogans strain Tarassovi VGNKI 4-inactivated Leptospira interrogans suspension of bacterial cells serogroup Grippotyphosa strain VGNKI-1, inactivated suspension of bacterial cells of the bacterium Leptospira interrogans serogroup Sejroe strain "Hardjo" and inactivated suspension of bacterial cells Leptospira interrogans serogroup Sejroe strain VGNKI-5, taken in a mixture in equal proportions with the final to the concentration of each leptospira strain is not less than 270 million microbial cells in 1 cm ^{3 of the} vaccine. 2. The vaccine according to claim 1, characterized in that it contains an adjuvant as a target additive. 3. The vaccine according to claim 2, characterized in that it contains saponin and / or aluminum hydroxide as an adjuvant.