RU2403063C1 - Inactivated combined vaccine against viral diarrhea, rota-, corona-virus diseases and escherichiosis of cattle - Google Patents

Abstract

FIELD: medicine, veterinary.

SUBSTANCE: invention relates to field of veterinary microbiology. Vaccine contains active substance and target additives. As active substance vaccine contains mixture of inactivated suspension of strain of cattle diarrhea virus SCV No 2336, family Flaviviridae, genus Pestivirus, inactivated suspension of strain of rotavirus SCV No 2337, family Reoviridae, genus Rotavirus; inactivated suspension of strain of coronavirus SCV No 2338, family Coronaviridae, genus Coronavirus, with activity of each of virus strains not lower than 10^6.5 TCD₅₀ in 1 cm³ of vaccine, and inactivated suspension of cells of bacteria Escherichia coli serovariants O9:K30, O78:K80, O115, O141 :K99 and O101:F41, taken in mixture in equal proportion with concentration not less than 20 billion microbial cells in cm³ of vaccine.

EFFECT: vaccine is superior to known vaccines in spectrum of specific protection and efficiency of activity for sensitive group of animals, duration of expressed immunity, safety for animals in calf and newborn calves, in stability of preservation of biological properties, including antigenic and immunogenic activity for 18 months (observation term).

3 cl, 8 tbl, 5 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, especially in newborn calves, accompanied by a violation of the functions of the gastrointestinal tract of varying severity.

Diseases of the gastrointestinal tract of young cattle (cattle) are widespread in all countries of the world and cause enormous economic damage. Mass enteritis and enterocolitis, clinically manifested by diarrhea, are recorded in 70-100% of newborn calves by the end of the first day after birth, and mortality of sick calves is 15-55% (Pavlov D.K. Diseases of the gastrointestinal tract in newborn calves. / / “Veterinary Life”, June, No. 11, 2006).

A distinctive feature of these diseases is the multiplicity of infectious agents that cause similar clinical manifestations of the disease. Mixed forms of infectious gastroenteritis are characterized by the intensive development of clinical signs and are caused by various associations of entropathogenic viruses, such as cattle diarrhea virus, rota- and coronaviruses, the virulence of which increases due to various adverse conditions of feeding and feeding. The course of viral infections, as a rule, is complicated by enterotoxigenic colibacteria, which are secreted by adult animals and enter the intestines of the newborn along with non-pathogenic colibacilli.

Since calves are infected with infectious pathogens in the first hours and days after birth, the basis of the specific prevention strategy for infectious gastrointestinal diseases is the active immunization of pregnant cows in order to provide passive protection of the offspring.

To date, several combination vaccines of various valencies against cattle gastroenteritis are known. However, their use is not always accompanied by a positive effect, mainly due to the imbalance of the antigenic composition and the low titer of specific antibodies in the blood of pregnant cows and in colostrum fed to newborn calves.

A combined vaccine against viral pneumogastroenteritis of cattle and calves is known containing adjuvant and inactivated suspensions of infectious rhinotracheitis viruses, viral diarrhea, rotavirus and coronavirus infection in a titer of 10 ^7.5 -10 ^8.5 TCD ₅₀ / _cm3, inactivated suspensions of parainfluenza-3 viruses and respiratory syncytial infection - in the titer of 10 ^6.5 -10 ^7.5 TCD ₅₀ / _cm3 taken in the vaccine in a volume ratio of 1: 1: 1: 1: 0.5: 0.5, respectively (Patent RU No. 2261111, A61K 39/295, publ. 09/27/2005).

However, the known combination vaccine does not provide immunological protection of animals against infectious gastroenteritis of bacterial etiology due to the absence of E. coli antigens in the vaccine. In addition, the vaccine strains of viruses that make up its composition do not provide immunity to different subtypes and main antigenic variants of epizootic strains of pathogens circulating on farms, and, as a result, the known vaccine is not effective enough.

Known associated vaccine against calf diarrhea containing ethanol-inactivated cells of the strains of Escherichia coli 09: K99, Escherichia coli 0138: K88, Escherichia coli GISK N 200, Salmonella tuphimurium GISK N 195, Salmonella dublin GISK N 196, Salmonella enteritidis Prussis vulgaris GISK N196 HIS N 198, Proteus mirabilis HIS N 199, Klebsiella pneumoniae HIS N 201, Klebsiella pneumoniae N HIS 202, phenol, isotonic sodium chloride solution and adjuvant aluminum hydroxide (Patent RU No. 2035916, A61K 39/125, publ. 17.06.93) .

A disadvantage of the known associated vaccine is its low efficiency due to the incompleteness of the antigenic composition of its constituent components, since the vaccine does not contain antigens of viral pathogens that cause massive outbreaks of cattle diarrheal diseases.

Also known is a formolthiomersal vaccine against calf Escherichiosis, which is a mixture of inactivated cells of enteropathogenic E. coli 08 strains: K43; 09, 015: K-14; 020, 026: K60; 041, 055, 078: K-80; 086: K-61: H32; 0115, 0117: K-62; 0119, 0141 and 0149 (Veterinary preparations. Handbook. - M .: Kolos, 1981, pp. 219-224).

However, the known vaccine does not protect animals with mixed forms of infectious gastrointestinal diseases, because It does not have specific activity against the main pathogens of a viral nature.

Known associated vaccine against rota-, corona-, herpesvirus and Escherichia diarrhea of newborn calves containing inactivated culture suspensions of strains of Bovine rotavirus "III-1", Bovine coronaviras "CM-1", Bovine Herpesvirus "TKA-VIEV-B2" with titers, respectively 107.0-107.8 TCD50 / ml, 105.0-105.8 TCD50 / ml, 107.6-108.0, inactivated cell suspensions of Ecsherichia coli strains synthesizing adhesins K99 and A20, with a concentration of 100-120 billion. m.k. in 1 ml of physiological saline, and a gel of aluminum hydroxide (Patent RU No. 2145236, A61K 39/295, publ. 10.02.2000).

The main disadvantage of the associated vaccine is the low antigenic and immunogenic activity of the vaccine strains, as well as the lack of immunity to escherichiosis occurring in the forms of colidiarrhea and colienterotoxemia.

The closest analogue is the combined inactivated vaccine against viral diarrhea, rota-, coronavirus disease, and calf disease “Kombovak-K”, which is made from strains of three viruses: rota-, corona- and viral diarrhea, protective antigens Escherichia; somatic 09, 078, 0115; capsular polysaccharide K 80, K 30; adhesive antigens 99, F-41, thermostable and thermolabile inactivated enterotoxins and is intended for the prevention of the diseases caused by them (Instructions for use of the inactivated combined vaccine against viral diarrhea, rota-, coronavirus disease, and calf escherichiosis “Combovac-K”, approved by the Department of Veterinary Medicine on 30.03. 2007).

A disadvantage of the known combination vaccine is the relatively low titer of infectious activity of the viral suspensions included in its composition, as a result of which the necessary amount of protective antigens against these diseases is not provided in the vaccine dose.

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 organs of the gastrointestinal tract, and thus ensure the safety of young animals and adult animal productivity.

Another objective is to obtain new production strains of viruses VD, PB and KB, 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 organs of the gastrointestinal tract, as well as to increase the stability, antigenic and immunogenic activity of the combined vaccine and create intense and prolonged immunity in vaccinated animals and born calves against viral diarrhea, company -, coronavirus disease and escherichiosis in cattle.

The technical result of the invention is achieved due to the use of new production strains of diarrhea virus, rota and coronaviruses of cattle with a wide antigenic spectrum in the composition of the vaccine, as well as by creating a synergistic composition of a polyvalent vaccine when combining new production strains of viruses with known vaccine strains of Escherichia.

The invention consists in the following.

The proposed inactivated combination vaccine against viral diarrhea, rotavirus, coronavirus disease and bovine Escherichiosis, containing the active substance and target additives, is characterized in that as the active substance it contains a mixture of inactivated suspension of the bovine diarrhea virus strain GKV No. 2336, family Flaviviridae, genus Pestivirus; inactivated suspension of a strain of rotavirus STB No. 2337, family Reoviridae, genus Rotavirus; inactivated suspension of a strain of coronavirus STB No. 2338, family Coronaviridae, genus Coronavirus; inactivated suspension of bacterial cells of Escherichia coli serovariants O9: K30, O78: K80, O115, O141: K99 and O101: F41, 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 diarrhea virus strain No. 2,336 of the cattle diarrhea virus included in the combined vaccine, the Flaviviridae family, the genus Pestivirus, the rotavirus strain of the GBV virus No. 2337, the Reoviridae family, the genus Rotavirus, and the coronavirus strain of the GBV No. 2,338, the Coronaviridae family, the new genus Coronavirus, are new first used in vaccine formulations.

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 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 1).

Table 1 Cross antigenic activity of the strain of ST-Bills No. 2336 virus VD 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 ° C.

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 - retains the original biological (antigenic) properties during successive passages in the culture of MDVC cells for 10 passages (observation period).

Pathogenicity - expressed.

Contagiousness - expressed.

Oncogenicity is absent.

The bovine rotavirus production strain GKV No. 2337 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 GKV participant No. 2337 and is characterized by the following features and properties.

Source of origin.

The source for a new production strain of cattle rotavirus is the K-88 virus strain (author's name), isolated in 1988 from the contents of the intestine of a calf with severe diarrhea, which passed 69 consecutive passages in transplanted cattle cell cultures.

Morphological properties.

GKV strain No. 2337 belongs to the Reoviridae family, the genus Rotavirus and has morphological characteristics characteristic of the causative agent of rotavirus infection of cattle: the virion is a rounded shape with a diameter of 60-80 nm, resembling the appearance of a wheel.

Antigenic properties.

GKV strain No. 2337 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 GKV No. 2,337 of cattle rotavirus leads to the formation of a humoral immune response to various strains and field isolates of cattle rotavirus (Table 2).

Table 2 Cross antigenic activity of a strain of ST-Bills No. 2337 of cattle rotavirus Ravavirus strain / isolate for pH The titer of virus-neutralizing antibodies in the blood serum of guinea pigs immunized with the STB strain No. 2,337 of rotavirus, M ± m ГКВ №2337 824.3 ± 46.1 K-88 406.2 ± 36.0 Isolate No. 2810 238.8 ± 26.4 Isolate No. P-47 219.4 ± 24.2 Isolate No. P-56 351.7 ± 34.2

Has antigenic affinity with rotaviruses isolated from other animal species.

Biotechnological characteristics.

The strain reproduces 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 7.5-8.5 lg TCD ₅₀ / cm ³ .

Recommended storage method.

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

Frequency of refreshment.

Once every three years in a culture of MDVC cells.

Additional information about the strain.

GKV strain No. 2337 is not contaminated by extraneous microflora. Tested with reference serum (GB) and ELISA.

Reactogenicity - the strain of ST-Bills No. 2337 does not have reactogenic properties.

Stability - retains the original biological (antigenic) properties during successive passages in the culture of MDVC cells for 10 passages (observation period).

Pathogenicity - expressed.

Contagiousness - expressed.

Oncogenicity is absent.

The bovine coronavirus production strain of bovine bacilli, No. 2338, was deposited in the State Virus Collection of the Institute of Virology of the Russian Academy of Medical Sciences, has the registration number of bills, No. 2338, and is characterized by the following features and properties.

Source of origin.

The source of the production of a new production strain of cattle coronavirus is the viral strain KV-90 (author's name), isolated in 1990 from the contents of the intestine of a calf with severe diarrhea in a culture of a kidney cell of a cow embryo, which passed 48 consecutive passages in cattle transplanted cultures.

Morphological properties.

GKV strain No. 2338 belongs to the Coronaviridae family, the genus Coronavirus, and has morphological characteristics characteristic of the causative agent of coronavirus infection of cattle: the virion is a round or oval shape with a diameter of 80-100 nm with club-shaped protrusions 12-24 nm long, forming a kind of solar corona. The virus genome is represented by single-stranded, fragmented RNA.

Biotechnological characteristics.

GKV strain No. 2338 propagates well in the primary culture of calf kidney cells (cow embryos) and MDVC cell culture. In these cell cultures, the virus accumulates in a titer of 6.5-7.5 lg TCD _{50 /} cm ³ .

Antigenic properties.

GKV strain No. 2338 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 pig strain No. 2,338 of bovine coronavirus cattle leads to the formation of a humoral immune response to various strains and field isolates of coronavirus cattle (table 3).

Table 3 Cross antigenic activity of a strain of STB No. 2338 of cattle coronavirus Strain / isolate of coronavirus for pH The titer of virus-neutralizing antibodies in the blood serum of guinea pigs immunized with a strain of STB No. 2338 coronavirus, M ± m ГКВ №2338 247.3 ± 41.8 KV-90 187.4 ± 36.6 Isolate No. 456 81.5 ± 12.8 Isolate No. 128 59.7 ± 6.7

Recommended storage method.

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

Frequency of refreshment: once every three years in the culture of MDVC cells.

Additional information about the strain: the strain is not contaminated by extraneous microflora. Tested with a clone crown.

Reactogenicity - does not possess reactogenic properties.

Stability - retains the original biological (antigenic) properties during successive passages in the culture of MDVC cells for 10 passages (observation period).

Pathogenicity - expressed.

Contagiousness - expressed.

Oncogenicity is absent.

As part of a combined vaccine against viral diarrhea, rotavirus, coronavirus disease and bovine Escherichiosis, inactivated viral suspensions of the strains of STB No. 2336, STB No. 2337 and STB No. 2338 are contained with an infectious activity titer of each virus of at least 10 ^6.5 TCD ₅₀ in 1 see ³ vaccines.

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 depending on the titer of the infectious activity of the viral suspensions, for example, with the titer of the infectious activity of strains of STB No. 2236, STB No. 2337 - 10 ^8.5-9.0 TCD ₅₀ / cm ³ and strain GKV No. 2338 -10 ^7.0-7.5 TCD ₅₀ / cm ³ viral suspensions are mixed in a ratio by volume of 2: 1: 1 (mixture No. 1); or with the titer of infectious activity of the strain of STB No. 2336 - 10 ^8.5-9.0 TCD ₅₀ / cm ³ , the strain of STB No. 2337 - 10 ^7.5-8.5 TCD ₅₀ / cm ³ , the strain of STB No. 2338 - 10 ^{6 5-7.0} TCD ₅₀ / cm ³ viral suspensions are mixed in a ratio by volume of 1: 1: 1 (mixture No. 2); or with the titer of infectious activity of the strain of STB No. 2336 - 10 ^7.5-8.5 TCD ₅₀ / cm ³ , the strain of STB No. 2337 - 10 ^8.5-9.0 TCD ₅₀ / cm ³ and the strain of STB No. 2338 - 10 ^{7 , 0-7.5} TCD ₅₀ / cm ³ viral suspensions are mixed in a ratio by volume of 4: 1: 1 (mixture No. 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 Escherichia coli strains of serovariants O9: K30, O78: K80, O115, O141: K99 and O101: F41 producing antigens: somatic O9, O78, O115; used for the manufacture of the Escherichia component of the combined vaccine; capsular polysaccharide K 80, K 30 and adhesive K 99, F-41 are known and used in various combinations in the manufacture of vaccines and sera against animal escherichiosis (see, for example, copyright certificate SU No. 1149467, RF patents No. 2043771, 2043772, 2070819 , 2053792, 2222592, etc.). However, we found that when combining known Escherichia strains with new production virus strains 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 Escherichia components of the vaccine.

The composition of the proposed vaccine inactivated cell suspensions of bacteria of strains of Escherichia coli serovariants O9: K30, O78: K80, O115, O141: K99 and O101: F41 are contained in equal proportions with a final concentration of at least 20 billion microbial cells in 1 cm ^{3 of the} vaccine.

Inactivation of Escherichia 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 acceptable 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 vaccine according to the invention preferably 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 vaccine is an inactivated combination vaccine against viral diarrhea, rotavirus, coronavirus disease and bovine Escherichiosis is a light red liquid, during storage a friable white precipitate forms, which easily breaks up into a homogeneous suspension when shaken.

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

The combined vaccine is administered to pregnant animals twice with an interval of 14-21 days, the first time 50-60 days before calving, the second time no later than 30 days before calving. The vaccine is administered intramuscularly to adult animals at a dose of 3 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 colostral immunity in newborn calves lasted up to 1.5 months.

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

Example 1. Obtaining an inactivated combination vaccine against viral diarrhea, rota-, coronavirus disease and Escherichia cattle.

The vaccine is prepared from new production strains of the diarrhea virus of the STB No. 2336, the rotavirus of the STB No. 2337, the coronavirus of the STB No. 2338 (viral component) and any known vaccine strains of bacteria Escherichia coli serovariants O9: K30, O78: K80, O115, O141: K99 and O01: O99 and O01: O99: O99 F41 (Escherichia component).

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

A. The step of obtaining the viral component of the combination vaccine.

The manufacture of the viral component of the combination vaccine includes separate steps for producing viral suspensions from the inoculum of strains of the diarrhea virus of GBV No. 2,336, rotavirus GBV No. 2,337 and coronavirus GBV No. 2,338; 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.

All virus strains used for the manufacture of the combination vaccine are grown in a transplanted MDVC calf kidney cell culture. For the cultivation of strains of rotavirus STB No. 2337 and coronavirus STB No. 2338, trypsin at a concentration of 5 μg / cm ^{3 is} added to the support medium.

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 titer of viruses reaches 10 ^6.5 -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 ^6.5 TCD ₅₀ / cm ³ .

Moreover, when the titer of infectious activity of strains of STB No. 2336, STB No. 2337 - 10 ^8.5-9.0 TCD ₅₀ / cm ³ and strain STB No. 2338 - 10 ^7.0-7.5 TCD ₅₀ / cm ³ viral suspensions are mixed in a volume ratio of 2: 1: 1 (mixture No. 1); or with the titer of infectious activity of the strain of STB No. 2336 - 10 ^8.5-9.0 TCD ₅₀ / cm ³ , the strain of STB No. 2337 - 10 ^7.5-8.5 TCD ₅₀ / cm ³ , the strain of STB No. 2338 - 10 ^{6 5-7.0} TCD ₅₀ / cm ³ viral suspensions are mixed in a ratio by volume of 1: 1: 1 (mixture No. 2); or with the titer of infectious activity of the strain of STB No. 2336 - 10 ^7.5-8.5 TCD ₅₀ / cm ³ , the strain of STB No. 2337 - 10 ^8.5-9.0 TCD ₅₀ / cm ³ and the strain of STB No. 2338 - 10 ^{7 , 0-7.5} TCD ₅₀ / cm ³ viral suspensions are mixed in a ratio by volume of 4: 1: 1 (mixture No. 3).

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

Bacterial strains of Escherichia coli of serovariants O9: K30, O78: K80, O115, O141: K99 and O101: F41 are taken, which in accordance with their antigenic formula produce somatic antigens O9, O78, O115; capsular polysaccharide antigens K 80, K 30 and adhesive antigens K 99, F-41.

The cultivation of Escherichia strains includes the preparation of matrix seedling with its subsequent sowing in the reactor.

For the preparation of matrix brood Escherichia culture of each strain is seeded with semi-liquid agar in a volume of 1-2 cm ³ in a separate bottle containing 80-100 cm ^{3 of} Hottinger broth. Crops are cultivated for 8-10 hours at a temperature of 37 ± 1 ° C. Grown cultures are checked for purity macroscopically and by microscopy of Gram stained smears. Pure cultures are each seeded separately in 10-liter bottles of Hottinger broth, filled not more than half the volume, and grown for 8-10 hours at a temperature of 37-38 ° C, periodically shaking. Checked for purity of growth, the matroe seedlings of each strain are seeded in a separate reactor in an amount of 5-10% of the medium volume, 40% glucose solution is added to a 0.2% concentration to the medium volume and grown for 10-14 hours at a temperature 37 ± 1 ° C with continuous aeration and stirring (air supply under pressure of 0.1-0.2 atm 50 l per hour). Co-cultivation of strains of serological variants O9: K30 and O78: K80 is allowed, taking into account their colicinogenicity. The brood seed strains are mixed in equal proportions.

Strains O115, O101: F41 and O141: K99 are grown in separate reactors. Changing the composition of groups is not allowed.

After growing, determine the concentration of microbial bodies in the culture, pH and purity of the culture.

The strains O101: F41 and O141: K99 grown in the culture reactors are separated in an ASG - ZM separator with a rotation frequency of 4500-5000 min ^-1 , the bacterial mass is resuspended in a reactor in phosphate-urea buffer to a concentration of 80-100 billion microbial bodies in 1 cm ³ and heated at a temperature of 60-65 ° C for 20 minutes. Supernatants are separated from the bacterial cells by centrifugation at a speed of 10,000-15,000 min ^-1 and checked for the content of adhesive antigens F41 and K99 in the diffuse precipitation reaction.

Next, the resulting cultures are combined in equal proportions, formalin diluted with physiological saline 1: 1 is added so that its concentration in the culture is 0.3%. Inactivation of cultures is carried out for 15-16 days at a temperature of 37 ± 1 ° C. The resulting mixture of inactivated cell suspensions is concentrated to obtain a back mass with a content of 100-200 billion microbial cells in 1 cm ³ .

B. Stage of combining the viral and Escherichia components of the combination vaccine.

Inactivated virus suspensions prepared in stage A are mixed in the prescribed ratio depending on the titer of the infectious activity of the viral suspension of each strain (mixture No. 1), mixed thoroughly. Then 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 the preservative sodium thiolate at a concentration of 1: 10000 are thoroughly mixed.

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

The viral and bacterial parts of the drug are mixed so that the concentration of viral antigens in the finished vaccine is at least 6.5 lg TCD ₅₀ , and Escherichia is at least 20 billion cubic meters. in 1 cm ³ .

Option 2

Inactivated virus suspensions prepared in stage A are mixed in the prescribed ratio depending on the titer of the infectious activity of the viral suspension of each strain (mixture No. 2), mixed thoroughly. Then 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 preservative sodium thiolate at a concentration of 1: 10000, mix thoroughly.

To inactivated bacterial cell suspension of a mixture of Escherichia strains prepared in stage B, add 6% GOA (20% of the total volume) and mix thoroughly. The viral and bacterial parts of the drug are mixed so that the concentration of viral antigens in the finished vaccine is at least 6.5 lg TCD ₅₀ , and Escherichia is at least 20 billion cubic meters. in 1 cm ³ .

Option 3

Inactivated virus suspensions prepared in stage A are mixed in the prescribed ratio, depending on the titer of the infectious activity of the viral suspension of each strain (mixture No. 3), mixed thoroughly. Then add 6% gel of aluminum hydroxide (GOA) in an amount of 30% of the total volume, mix thoroughly.

To inactivated bacterial cell suspension of a mixture of Escherichia strains prepared in stage B, add 6% GOA (30% of the total volume) and mix thoroughly.

The viral and bacterial parts of the drug are mixed so that the concentration of viral antigens in the finished vaccine is at least 6.5 lg TCD ₅₀ , and Escherichia is at least 20 billion cubic meters. in 1 cm ³ .

The obtained variants of the inactivated combined vaccine against viral diarrhea, rota-, coronavirus diseases, and bovine Escherichiosis have a component composition shown in Table 4

Table 4 The content of ingredients in the liquid mixture, vol.% Ingredient Titer, lg TCD ₅₀ / cm ³ The content in the liquid mixture, vol.% Option 1 Option 2 Option 3 VD virus 7.5-8.5 40 8.5-9.0 thirty twenty Rotavirus 7.5-8.5 twenty 8.5-9.0 fifteen 10 Coronavirus 6.5-7.0 twenty 7.0-7.5 fifteen 10 Escherichia strain cell suspension - 19.94 19.94 10.0 GOA (6% gel) - twenty twenty thirty Saponin - 0.05 0.05 - Mertiolate Sodium - 0.01 0.01 -

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

To determine the antigenic and immunogenic activity of the combination vaccine according to the invention, laboratory animals are used as an experimental model, while to assess the antigenic activity of the viral components of the vaccine, guinea pigs weighing 350-400 g are used, to determine the antigenic activity of adhesive antigens of E. coli, rabbits weighing 3- 3.5 kg, to assess the immunogenic activity against somatic antigens of E. coli - white mongrel mice weighing 18-20 g.

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 of 1.0 cm ³ . In this case, the vaccine is administered intramuscularly to guinea pigs, and subcutaneously 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 viruses VD, RV and KB. 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 dilution 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 E. coli adhesive antigens in a vaccine is evaluated in an agglutination reaction (RA) by the titer of antibodies in the blood serum of vaccinated rabbits.

To assess the immunogenic activity of the vaccine in relation to somatic antigens of E. coli, 2 groups of white mice of 20 animals each are formed. Animals of the control group are immunized twice with an interval of 10 days. The vaccine is administered subcutaneously at a dose of 0.1 cm ³ . Animals of the control group are not immunized.

After 16-18 days. after repeated injection of the vaccine, the mice are infected with an intraperitoneally titrated lethal dose of two strains of E. coli serogroups O78 and O115. For each serogroup strain, 10 vaccinated and 10 control animals are used.

The immunogenic activity of the vaccine in relation to somatic antigens of E. coli is evaluated by the number of surviving animals in the experimental group.

The results of the study are presented in Table 5, which indicate that the combined vaccine against viral diarrhea, rota-, coronavirus diseases, and bovine escherichiosis 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 viral components of the drug in high titer: to the VD virus - 1: 222.9, to rotavirus - 1: 294.0, to coronavirus - 1: 207.9 ; to adhesive antigens of Escherichia - from 1: 540.5 to 1: 720.0, the immunogenic activity of the vaccine is 90-100%.

Animals of the control group throughout the experiment were seronegative for viruses VD, PB, KB and Escherichia.

Table 5 Antigenic activity of the combined vaccine against viral diarrhea, rota-, coronavirus diseases, and bovine Escherichiosis in experimental models Live group Guinea pig serum antibody titer Batch number Antibody titer in rabbit serum to adhesive. Ag Immunogenic activity of the vaccine,% b / m survivors The dose of the drug, cm ³ The titer of neutralizing antibodies, M ± m VD RV KB O78 O115 I 0.25 28.8 ± 2.1 35.2 ± 8.7 86.4 ± 5.7 one 540.5 ± 25.4 90 90 II 0.5 222.9 ± 23.2 294.0 ± 28.5 207.9 ± 24.5 2 720.0 ± 68.3 one hundred one hundred III 1,0 104.0 ± 14.8 157.6 ± 16.4 181.0 ± 19.2 3 695.5 ± 42.2 90 one hundred the control - 0 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 are used, and the Escherichia component is used in rabbits and white mice.

For this, the vaccine is administered after 6, 12 and 18 months. storage at a temperature of 4 ° C once: guinea pigs at a dose of 0.5 cm ³ , rabbits at a dose of 1.0 cm ³ . 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, and of the Escherichia component in RA by the absence of a decrease in antibody titer below the established level (1:16 and 1: 200, respectively) when the vaccine is stored at + 4 ° C for 18 months.

The test results are presented in table.6.

Table 6 Change in antigenic activity of the combined vaccine during storage Storage time The titer of antibodies in the blood serum of vaccinated laboratory animals to the pathogen VD RV KB Esherichia When made. 222.9 ± 23.2 294.0 ± 28.5 207.9 ± 24.5 720.0 ± 68.3 6 months 207.9 ± 24.5 274.4 ± 18.6 128.0 ± 16.9 649.8 ± 52.7 12 months 157.6 ± 16.4 181.0 ± 16.7 90.5 ± 13.4 527.8 ± 34.1 18 months 119.4 ± 21.2 111.4 ± 14.5 59.7 ± 10.9 324.9 ± 25.6

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

The test is carried out on pregnant cows and heifers in farms with a high level of gastrointestinal diseases of newborn calves, dysfunctional due to viral diarrhea, rota-, coronavirus diseases and bovine escherichiosis.

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 30 days before calving.

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 efficiency is assessed by the level of virus-neutralizing antibodies in the blood serum of cows and by Escherichia - by the level of antibodies in RA to the Escherichia adhesive antigens.

The test results are presented in table.7.

Table 7 Evaluation of the activity of the vaccine against viral diarrhea, rota-, coronavirus diseases, and bovine Escherichiosis in an experiment on cows Duration of studies, months after vaccination The serum antibody titer to the pathogen VD RV KB E.coli Experienced group Before vaccination 18.9 ± 6.4 23.6 ± 2.9 29.0 ± 5.6 61.7 ± 15.1 one 476.7 ± 30.1 961.3 ± 46.2 1351.6 ± 44.0 649.8 ± 34.6 2 512.0 ± 21.3 1229.0 ± 61.4 1792.6 ± 69.1 492.5 ± 31.2 four 494.5 ± 32.3 684.1 ± 38.1 749.6 ± 48.2 373.2 ± 28.9 6 247.3 ± 41.8 394.0 ± 28.9 430.5 ± 38.1 324.9 ± 24.7 8 163.1 ± 14.5 238.8 ± 36.4 349.7 ± 35.6 282.8 ± 18.3 12 76.8 ± 20.2 82.9 ± 22.6 100.4 ± 21.2 162.5 ± 15.8 Control group - 17.5 ± 3.9 16.2 ± 8.9 32.9 ± 14.7 83.2 ± 21.4

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. Evaluation of colostral immunity in newborn calves.

In the experiments, 7-14-day-old calves born to cows immunized with the combination vaccine of 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 virus-specific antibodies in the blood serum of all experimental and control animals and to Escherichia by the level of antibodies to Escherichia adhesive antigens. The test results are presented in table.8.

Table 8 Evaluation of the colostral immunity of the vaccine against viral diarrhea, rota-, coronavirus diseases, and bovine Escherichiosis in an experiment on calves Age of newborn calves, weeks The serum antibody titer to the pathogen VD RV KB E.coli Experienced group one 494.5 ± 32.3 1097.5 ± 36.6 1260.7 ± 34.8 527.8 ± 38.2 2 238.9 ± 23.4 749.6 ± 31.2 803.4 ± 25.1 373.2 ± 28.9 four 163.1 ± 21.4 461.4 ± 28.6 476.7 ± 30.1 114.9 ± 11.2 6 107.6 ± 24.1 100.4 ± 16.9 84.4 ± 18.5 57.4 ± 14.5 Control group one 19.5 ± 5.9 21.2 ± 8.5 29.9 ± 10.2 0 2 0 0 12.4 ± 8.6 0 four 0 0 0 0 6 0 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 and Escherichia, which persisted throughout the observation period.

Thus, the experiments show that the inactivated combination vaccine against viral diarrhea, rotavirus, coronavirus disease, and bovine Escherichiosis in accordance with the invention has a decisive advantage over other known vaccines in the spectrum of specific protection and efficacy for a susceptible group of animals, the duration of intense immunity, harmlessness to pregnant animals, on 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 viral diarrhea, rotavirus, coronavirus disease and bovine Escherichiosis, containing the active substance and target additives, characterized in that as the active substance it contains a mixture of inactivated suspension of the strain of bovine diarrhea virus of bovine animals No. 2336, family Flaviviridae, genus Pestivirus, inactivated suspension of a strain of rotavirus STB No. 2337, family Reoviridae, genus Rotavirus; inactivated suspension of the strain of coronavirus STB No. 2338, family Coronaviridae, genus Coronavirus, with the activity of each of the viral strains of at least 10 ^6.5 TCD ₅₀ in 1 cm ^{3 of the} vaccine, and inactivated suspension of bacterial cells Escherichia coli serovariants O9: K30, O78: K80, O115, O141: K99 and O101: F41 taken in a mixture in equal proportion with a concentration of at least 20 billion 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.