RU2546247C2 - Vaccine against plague, adenovirus infections, parvovirus and coronavirus enteritis, leptospirosis and rabies of dogs - Google Patents

Abstract

FIELD: veterinary medicine.

SUBSTANCE: vaccine against plague, adenovirus infections, parvovirus and coronavirus enteritis, leptospirosis and rabies of dogs is proposed, containing the active ingredient and targeted additives, characterised in that as the active ingredient it comprises in 1 dose of vaccine the mixture of suspension of attenuated strain of canine distemper virus SCV No. 2313 family Paramyxoviridae, genus Morbillivirus with a titre of at least 10^3.5 TCD₅₀; suspension of attenuated strain of adenovirus of dogs of type 2 SCV No. 2311, family Adenoviridae, genus Mastadenovirus with a titre of at least 10^3.0 TCD₅₀; suspension of attenuated strain of canine parvovirus of type 2 SCV No. 2312, family Parvoviridae, genus Parvovirus with a titre of at least 10^3.0 HAU; suspension of attenuated strain of canine coronavirus SCV No. 2314, family Coronaviridae, genus Coronavirus with a titre of at least 10^3.0 TCD₅₀; inactivated suspension of strain of rabies of dogs ERA-CB-M20, family Rhabdoviridae, genus Lyssavirus in the amount of 1 ME, inactivated suspensions of leptospira of serogroups Icterohaemorrhagiae and Canicola taken in the mixture in equal proportions with the final concentration of each strain of at least 3×10⁸ inactivated microbial cells in 1 dose of the vaccine.

EFFECT: invention is characterised by higher antigenic and immunogenic potency, safety, ability to create colostral immunity of high intensity, high stability in storage, high specificity in relation to different subtypes and main antigenic variants of epizootic strains of pathogens circulating in the outer environment in the territory of Russia and the CIS countries, the vaccine is environmentally friendly, does not cause dissemination of pathogenic agents.

4 cl, 13 tbl, 8 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 for the specific prevention of infectious diseases of dogs. Plague, infectious hepatitis, infectious laryngotracheitis, parvovirus and coronavirus enteritis, leptospirosis and rabies are the most common and dangerous infectious diseases. dogs. These diseases are found everywhere in dogs of different breeds and ages. Plague and parvovirus enteritis are highly contagious diseases, mortality from which for puppies can reach 100%. Infectious hepatitis and infectious laryngotracheitis, caused respectively by type 1 and type 2 adenoviruses, infect dogs of any age and can cause severe damage to the liver and respiratory tract, leading to the death of the animal. Coronavirus enteritis is most dangerous for puppies in the first months of life. Rabies and leptospirosis are zooanthroponoses and pose a serious danger not only to dogs, but also to their owners. To date, several combination vaccines of various valencies against these infections are known, but their use is not always accompanied by a positive effect, because they used mainly inactivated strains of viruses, which are significantly inferior in immunogenicity to live attenuated strains. In addition, inactivated vaccines, unlike live ones, are not able to stimulate cellular immunity, which plays an important role in protecting animals from infection. Also, the immunogenicity of vaccines used for specific prophylaxis of infectious diseases of dogs is greatly influenced by the system of cultivation of viral strains (especially for parvovirus dogs). The use of heterologous cultures, for example, cat cell cultures for producing dog parvovirus, significantly reduces the immunogenicity of vaccine parvovirus. Known vaccine against plague, infectious hepatitis and parvovirus enteritis carnivores, containing a lyophilized mixture of a suspension of the vaccine strain "VNIIVVmM-88" plague carnivores with a titer of infectious activity of 3.5-4.5 lg TPD 50 / cm ³ suspension of the vaccine strain "Cornell-2 "infectious hepatitis in dogs with a titer of infectious activity of 5.5-6.5 lg TPD 50 / cm ³ , a suspension of vaccine strain A" of panleukopenia of cats with a titer of infectious activity of 3.5-4.5 lg ID 50 / cm ³ and hemagglutinating activity 1: 32-1: 512 and a drying medium based on lactose, sucrose, s orbit and gelatin (patent RU No. 2154496, A61K 39/295, publ. 20.08.2000). 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 for dogs against infectious laryngotracheitis, coronavirus enteritis, rabies and leptospirosis. In addition, the vaccine strain “A” of feline panleukopenia included in its composition has insufficient immunogenicity and does not provide the necessary degree of protection for dogs against virulent strains of the dogs parvovirus enteritis virus. Known vaccine "Tetravac" for the prevention of plague, infectious hepatitis, adenovirus infections and parvovirus enteritis in dogs, including an inactivated suspension of the strain Mammaliadenovirus Adenovirus canis-2 "Ada" with a titer of infectious activity of 10 ^4.5 -10 ^5.0 TCD ₅₀ / cm ³ inactivated strain slurry Virus panleucopenia felline "Gannibal" with a titer of infectious activity of 10 ^4.0 -10 ^5.0 ID ₅₀ / cm ³ and to the activity in the RGA 1 ° C 64-256 Gai / cm ^3, a suspension Distem strain _50/0 , 5 cm ³ aluminum hydroxide, 25% gelatose solution, 50% sorbitol solution, distilled water and Hanks solution ( patent RU No. 2030917, A61K 39/295, publ. 03.20.1995). However, the Tetravac vaccine does not provide immunological protection for dogs against rabies, coronavirus enteritis and leptospirosis due to the absence of the corresponding antigens in the vaccine. In addition, the known vaccine is not effective enough, since the inactivated strains of dog type 2 adenovirus and feline panleukopenia virus included in its composition do not provide the formation of cellular immunity. Known vaccine "Hexacanivac" for specific prophylaxis of carnivore plague, infectious hepatitis, adenovirus, parvovirus enteritis and leptospirosis of dogs, containing a culture virus suspension of the strain Distemper canine "EPM" with a titer of infectious activity 10 ³ -10 ⁴ TCD ₅₀ / _0.5 cm ³ , inactivated culture virus suspension of the strain Mammaliadenovirus Adenovirus canis 2 "Ada" with a titer of infectious activity 10 ^4,5 -10 ⁵ TCD ₅₀ / cm ³ inactivated culture virus suspension of the strain Virus panleucopenia Felline "Hannibal" with a titer of infectious activity 10 ⁴ -1 0 ⁵ ID ₅₀ / cm ^3, and activity in the RGA 1: 64-1: 256 Gai / cm ^3, Leptospira Canicola serogroup VGNKI-3 strain and strain VGNKI serogroup Icterohaemorrhagiae-2 in equal proportions at a concentration of 300-500 million cells / 0. , 2 cm ³ , adjuvant - aluminum hydroxide and serum protein, stabilizer - solutions of gelatose and sorbitol (patent RU No. 2045960, А61К 39/295, publ. 10/20/1995). Also known is the associated vaccine "Biovak" against carnivorous plague, parvovirus enteritis, infectious hepatitis, adenovirus and leptospirosis of dogs, containing in one vaccination dose (2 ml) lyophilized virus holding a liquid virus strain of canine distemper EPMM with a stabilizer at a titer of at least 2.2 lg TCD _50, the suspension was adsorbed inactivated culture strain parvovirus carnivores "Hercules" at a titer of at least 6 log ₂ Gai (prior to inactivation), inactivated viral suspension of the strain Mammaliadenovirus Adenovirus canis - 2 "Ada" in a titer of at least 4 lg TCD ₅₀ and a mixed suspension of leptospira serogroups Canicola-3 and Icterohaemorrhagiae VGNKI-2, taken in equal proportions of microbial cells, with a total concentration of 200-400 million m cells The dry component of the vaccine is a lyophilized virus-containing liquid of the strain of the carnivore plague virus, mixed with the remaining liquid components of the vaccine immediately before use (patent RU No. 2150296, A61K 39/295, publ. 10.06.2000). The disadvantage of the Hexacanivac and Biovac vaccines is the use of inactivated virulent strains of dog adenovirus and dog parvovirus, which are significantly inferior to live attenuated strains of these viruses in immunogenicity. In addition, the use of cat cell cultures for the cultivation of dog parvovirus significantly reduces the immunogenicity of the vaccine strain of parvovirus, because when passaged in a heterologous cell culture, serious changes in the antigenic structure of virions occur, which leads to differences in the antigenic structure of the vaccine virus and isolates circulating in nature. A significant drawback of these vaccines is that they do not create immunity against coronovirus enteritis and rabies in dogs, which requires the additional use of monovaccines. Patent RU 2400248 describes combination polyvalent vaccines against dog pathogens containing, in one embodiment, a weakened Snyder Hill strain of dog plague virus (CD), a weakened strain of Manhattan dog adenovirus type 2 (CAV-2), a weakened strain of the parainfluenza virus NL-CPI-5 dogs (CPI), attenuated strain of dogs NL-35-D parvovirus (CPV), where the specified virus CD, CAV-2, virus CPI and CPV are in the range from 10 ² to 10 ⁹ TCID ₅₀ (infectious dose with 50% cytopathic effect in tissue culture) on the dose, inactivated preparation of a strain of dogs NL-18 coronavirus (CCV) from whole whether partial cells, wherein said CCV is present in an amount of at least about 100 relative units per dose, cell preparation of Leptospira serovars of Leptospira bratislava, Leptospira canicola, Leptospira grippotyphosa, Leptospira icterohaemorrhagiae and Leptospira pomona, and a carrier. This vaccine composition is the closest analogue (patent RU 2400248, A61K 39/295, publ. 09/27/2010). The well-known combined multivalent vaccine contains live attenuated viruses of dog adenovirus and parvovirus, however, the closest analogue is characterized by insufficient antiepizootic efficacy due to the fact that the strains used for the manufacture of the multivalent vaccine have antigenic and immunobiological differences compared with similar characteristics of epizootic pathogens circulating in Russia and countries CIS. In this regard, the objective of the invention is the creation of a safe and highly immunogenic vaccine against major infectious diseases of dogs, containing antigenically and immunogenically balanced viral and leptospirosis antigens, which provide the formation of stable immunity against the plague virus, adenoviruses, parvo and coronaviruses of dogs, rabies virus and leptospira. Another objective is to obtain new production strains of carnivore plague virus, dog adenoviruses, dog parvo and coronaviruses and rabies virus, which have high antigenic and immunogenic activity, stably retain their properties and are suitable for creating combined vaccines of different valencies. The technical result of the invention is to expand the arsenal of combined vaccines against major infectious diseases of dogs, to increase the stability, antigenic and immunogenic activity of the vaccine, as well as to increase the immunity in vaccinated animals by obtaining virions with a full spectrum of antigens and providing the possibility of stimulating cellular immunity. The technical result of the invention is achieved by using new vaccine strains of dog plague viruses, dog adenoviruses, dog parvo and coronaviruses, rabies virus and known leptospira strains with high antigenic and immunogenic activity in the vaccine, and the formation of a synergistic polyvalent vaccine composition when combining new production virus strains with known leptospira vaccine strains. The essence of the invention is as follows: the proposed combination vaccine against plague, adenovirus infections, parvovirus and coronavirus enteritis, leptospirosis and rabies dogs, containing the active substance and targeted additives, characterized in that as an active substance it contains a mixture of an attenuated strain suspension in 1 dose of the vaccine virus of the plague of carnivores Bills No. 2313, family Paramyxoviridae, genus Morbillivirus with a titer of at least 10 ^3.5 TCD ₅₀ ; suspensions of an attenuated strain of adenovirus dogs of type 2 STB No. 2311, family Adenoviridae, genus Mastadenovirus with a titer of at least 10 ^3.0 TCD ₅₀ ; suspensions of an attenuated strain of parvovirus of dogs of type 2 GKV No. 2312, family Parvoviridae, genus Parvo virus with a titer of at least 10 ^3.0 GAE; suspension of attenuated strain of coronavirus of dogs STB No. 2314, family Coronaviridae, genus Coronavirus with a titer of at least 10 ^3.0 TCD ₅₀ ; inactivated suspension of the strain of rabies virus EPA - CB-M20, family Rhabdoviridae, genus Lyssavirus in an amount of 1 ME; inactivated suspensions of leptospira serogroups Icterohaemorrhagiae and Canicola, taken in a mixture in equal proportion with the final concentration of each strain of at least 3 × 10 ⁸ inactivated microbial cells in 1 dose of the vaccine. The vaccine strain GVB No. 2313, the strain of dog adenovirus of the 2nd type GKV No. 2311, the strain of the parvovirus of dogs of the 2nd type GKV No. 2312, the strain of coronavirus of dogs GKV No. 2314, the strain of rabies virus ERA SV-20M included in the combination vaccine are new production strains and are 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 term "effective amount" means the amount of active substance that, when administered to dogs and / or puppies, provides the induction of a specific immune response of a sufficient level in the animal body, followed by warning against diseases and symptoms of diseases to be prevented. The production strain of the plague virus of carnivorous STB No. 2313 was deposited in the State Virus Collection of the Institute of Virology RAMS, has the registration number of the STB depositor No. 2313 and is characterized by the following features and properties.

Source of origin: The source of the new production strain of the carnivore plague virus is virus strain No. 37 (author's name), isolated in 1988 from the tissue of a spleen puppy that has been adapted to the transplanted culture of African green monkey kidney cells (Vero). Morphological properties: The strain of the virus of the plague of carnivorous STB No. 2313 belongs to the family Paramyxoviridae, the genus Morbillivirus. An electron microscopic examination using the negative contrast method of virus-containing culture material revealed virions characteristic of the carnivore plague virus: pleomorphic particles 100-300 nm in size and consisting of a nucleocapsid of spiral symmetry and a lipoprotein membrane. Antigenic properties: The strain of the virus of the plague of carnivorous STB No. 2313 shows high antigenic and immunogenic activity and has a wide antigenic spectrum. Information on the antigenic and protective activity of the GKV strain No. 2313 is presented in Tables 1 and 2. The sera of animals immunized with other strains of the carnivorous plague virus suppress the cytopathic activity of the GKV strain No. 2313 (Table 1). Once the introduction of the strain GKV No. 2313 of the virus of carnivorous plague at a dose of 10 ³ TCD ₅₀ thorzofretkam, protects them during control infection with 100 LD ₅₀ strain S. hill (table 2)

Table 1 Cross antigenic activity of a strain of the virus of the plague of carnivorous STB No. 2313 Reference serums to strains of VChP Serum neutralizing antibody titer (log2) Reference serum titer in PH with a strain of STB No. 2313 ГКВ №2313 - 9 Onderstepoort 8 9 Rockborn 7 7 S. hill 8 7 EPM 9 8

Table 2. The results of the control infection of thorzofretok vaccinated with a strain of the plague virus of carnivorous STB No. 2313 Group No. Qty Vaccination Control Control results py well done infection infection one 8 ГКВ №2313 S. hill Animals clinically 10 ³ TCD ₅₀ 100LD ₅₀ are healthy Typical clinical signs of canine distemper (rhinitis, conjunctivitis, refusal to feed), death 4-6 days after infection Control unvaccinated S. hill 100LD ₅₀ 2 8

Biotechnological characteristics: The strain of the virus of the plague of carnivorous STB No. 2313 reproduces well in the transplanted culture of kidney cells of the African green monkey (Vero) and accumulates in the titer of 6.5-7.0 lg TCD ₅₀ / cm ³ . Recommended storage method:

In a lyophilized state at a temperature not exceeding minus 600 ° С.

Frequency of refreshment:

Once every three years in a transplanted kidney cell culture African green monkey (Vero).

Additional information about the strain:

The strain of the plague virus of carnivorous STB No. 2313 is free of extraneous microflora.

Reactogenicity - the strain of the virus of the plague of carnivorous STB No. 2313 does not have reactogenic properties.

Stability preserves the original biological properties during successive passages in a Vero cell culture for 10 passages (observation period).

Pathogenicity - attenuated.

Contagiousness is absent.

Oncogenicity is absent.

The production strain of dog adenovirus type 2 STB No. 2311 was deposited in the State Virus Collection of the Institute of Virology RAMS, has the registration number of the STB Depositor No. 2311 and is characterized by the following features and properties.

Source of Origin:

The source of the new production strain of type 2 dog adenovirus is the viral strain No. 46 (author's name), isolated in 1989 from lung tissue of a fallen puppy, which is adapted to the transplanted culture of dog kidney cells (MDSK).

Morphological properties:

The strain of adenovirus dogs of type 2 STB No. 2311 belongs to the family Adenoviridae, genus Mastadenovirus. Electron microscopic examination using the method of negative contrasting of virus-containing culture material revealed virions characteristic of dog adenovirus: particles in the form of an icosahedron with a size of 70-100 nm and consisting of 252 subunits (capsomeres), from the tops of which club-shaped shoots with a diameter of 2 nm and a length of 20-50 nm

Antigenic properties:

The strain of adenovirus dogs of type 2 ST-Bills No. 2311 has a high cross antigenic and immunogenic activity in relation to other strains and field isolates of adenoviruses of dogs of the 1st and 2nd type. The introduction of the GKV strain No. 2311 into animals leads to the formation of a humoral immune response to various strains and field isolates of adenovirus dogs of types 1 and 2 (Table 3).

Table 3 Cross antigenic activity of a strain of dogs adenovirus GKV No. 2311 Adenovirus pH strain / isolate Geometric average titer of virus-neutralizing antibodies in the blood serum of puppies immunized with the HBV strain No. 2311 of adenovirus (Iog2) ГКВ№2311 8.2 VGNKI 7.8 Ada 8.4 Isolate No. 344 9 Isolate No. 1129 7.2

Biotechnological characteristics: GKV strain No. 2311 propagates well in primary and transplantable cultures of dog kidney cells. In these cell cultures, the virus accumulates in a titer of 5.5-6.5 lg TCD ₅₀ / cm ³ .

Recommended storage method:

In a lyophilized state at a temperature not exceeding minus 600 ° С.

Frequency of refreshment:

Once every three years in a transplanted dog kidney cell culture (MDCK).

Additional information about the strain:

The strain of dogs adenovirus type 2 GKV No. 2311 is free from extraneous microflora.

Reactogenicity - a strain of adenovirus of dogs of the 2nd type of STB No. 2311 does not have reactogenic properties.

Stability - preserves the initial biological properties during successive passages in a culture of MDSK cells for 10 passages (observation period).

Pathogenicity - attenuated.

Contagiousness is absent.

There is no oncogenicity. The production strain of parvovirus of dogs of the 2nd type of GBV No. 2312 was deposited in the State Virus Collection of the Institute of Virology RAMS, has the registration number of the GBV Depositor No. 2312 and is characterized by the following symptoms and properties.

Source of Origin:

The source of the production of a new production strain of parvovirus of dogs of type 2 is the virus strain No. 23 (author's name), isolated in 1988 from the feces of sick dogs with diarrheal syndrome, which is adapted to the transplanted culture of dog kidney cells (MDSK). The virus multiplies in the epithelial cells of the small intestine and at the level of 60-85 passage does not cause disease in 8-14 week old puppies with oral, subcutaneous and intramuscular injection.

Morphological properties:

The strain of parvovirus dogs of the 2nd type of ST-Bills No. 2312 belongs to the family Parvoviridae, genus Parvovirus. Electron microscopic examination using the method of negative contrasting of virus-containing culture material revealed virions characteristic of dog parvovirus: non-shell particles of icosahedral symmetry with a size of 20-25 nm.

Antigenic properties:

The strain of parvovirus of dogs of type 2 GKV No. 2312 has a high cross antigenic and immunogenic activity in relation to other strains and field isolates of parvoviruses of dogs of type 2. The introduction of an STB strain No. 2312 into animals leads to the formation of a humoral immune response to various strains and field isolates of dog parvovirus (Table 4).

Tab. four Cross antigenic activity of the parvovirus strain of dogs ГКВ№2312 Parvovirus strain / isolate for rtga The geometric mean titer of antihemagglutinins in the blood serum of puppies immunized with the STB strain No. 2312 (log ₂ ) ГКВ №2312 9.5 Isolate No. 1367 9.5 Isolate No. 1446 11.1 Isolate No. 1490 10.6

Biotechnological characteristics:

GKV strain No. 2312 propagates well in primary and transplantable cultures of dog kidney cells. In these cell cultures, the virus accumulates in a titer of 105-4 * 10 ⁵ GAU / cm ³ .

Recommended storage method:

In a lyophilized state at a temperature not exceeding minus 600 ° C.

Frequency of refreshment:

Once every five years in a transplantable dog kidney cell culture (MDCK).

Additional information about the strain:

GKV strain No. 2312 is free from extraneous microflora.

Reactogenicity - the strain of ST-Bills No. 2312 has no reactogenic properties.

Stability - preserves the initial biological properties during successive passages in a culture of MDSK cells for 10 passages (observation period).

Pathogenicity - attenuated.

Contagiousness is absent.

Oncogenicity is absent.

The production strain of coronavirus of dogs ГКВ №2314 was deposited in the State collection of viruses of the Institute of Virology RAMS, has the registration number of the participant ГКВ №2314 and is characterized by the following features and properties.

Source of Origin:

The source of the new production strain of dog coronavirus is the virus strain No. 49 (author's name), isolated in 1989 from feces of puppies with diarrheal syndrome, which is adapted to the transplanted culture of dog kidney cells (MDSK).

Morphological properties:

GKV strain No. 2314 belongs to the family Coronaviridae, the genus Coronavirus. An electron microscopic examination using the negative contrast method of virus-containing culture material revealed virions characteristic of dog coronavirus: particles of 80-100 nm in size, consisting of a nucleocapsid of spiral symmetry and a lipoprotein membrane, with characteristic club-shaped protrusions about 20 nm in length.

Antigenic properties:

GKV strain No. 2314 has a high cross antigenic and immunogenic activity in relation to other strains and field isolates of dog coronaviruses.

The introduction of an STB strain No. 2314 into animals leads to the formation of a humoral immune response to various field isolates of dog coronaviruses (Table 5).

Table 5 Cross antigenic activity of strain GKV No. 2314 of the corona virus of dogs Strain / isolate of coronavirus for pH Geometric mean titer of virus-neutralizing antibodies in the blood serum of puppies immunized with AHB No. 2311 adenovirus (log2) ГКВ №2314 6.25 Isolate No. 344 5.75 Isolate No. 1129 5.5 Isolate No. 344 6.75

Biotechnological characteristics:

GKV strain No. 2314 propagates well in primary and transplantable cultures of dog kidney cells. In these cell cultures, the virus accumulates in a titer of 5.5-6.0 lg TCD ₅₀ / cm ³ .

Recommended storage method:

In a lyophilized state at a temperature not exceeding minus 600C.

Frequency of refreshment:

Once every three years in a transplanted dog kidney cell culture (MDCK).

Additional information about the strain:

GKV strain No. 2314 is free from extraneous microflora.

Reactogenicity - strain GKV No. 2314 does not have reactogenic properties.

Stability - preserves the initial biological properties during successive passages in a culture of MDSK cells for 10 passages (observation period).

Pathogenicity - attenuated.

Contagiousness is absent.

Oncogenicity is absent.

The production strain of Era rabies virus - SV-20M was deposited in the State Collection of Microorganisms of the State Research Institute for Control, Standardization and Certification of Veterinary Medicines - Center for the Quality of Veterinary Medicines and Feed (FGU-VGNKI) and is characterized by the following features and properties.

Source of origin.

 The strain EPA - CB-M20 originates from the EPA strain (Abelse M, 1964) and is obtained by adapting it to BHK-21 cells and isolating the highly immunogenic and reproductive biological variant of the virus from the EPA strain. The strain EPA - CB-M20 belongs to the family Rhabdoviridae, the genus Lyssavirus.

An electron microscopic examination using the negative contrast method of virus-containing culture material revealed bullet-shaped virions of 170-380 nm in length and 65-70 nm in diameter, consisting of a spiral-circular cylindrical nucleocapsid and membrane, characteristic of rabies virus.

Biological properties: Strain Ep - CB-M20 is intensively propagated in the culture of VNK-21, pathogenic with intracerebral and peripheral infection methods for laboratory animals: mice, Syrian hamsters and guinea pigs. The titer of the strain during intracerebral infection of mice weighing 7-8 g is 8-9.0 lg LD ₅₀ / ml

Antigenic properties:

Strain Ep - SV-M20 has high antigenic and immunogenic activity for laboratory animals, dogs, Arctic foxes.

Recommended storage method:

In a lyophilized state at a temperature not exceeding minus 600 ° C.

Frequency of refreshment:

Once every ten years in the BHK-21 cell system, pig kidney cells.

Additional information about the strain:

The strain Ep - SV-M20 is free from extraneous microflora, not contaminated with foreign viruses.

Reactogenicity - EPA strain - CB-M20 does not have reactogenic properties.

Stability - retains the original biological properties during successive passages in the cell culture of BHK-21 for 10 passages (observation period). Pathogenicity - pathogenic with intracerebral infection of white mice, weakly pathogenic with peripheral methods of infection of white rats and guinea pigs and non-pathogenic with peripheral methods of infection of rabbits, arctic foxes, cats.

Contagiousness is absent.

Oncogenicity is absent.

The industrial strains of Leptospira interrogans serogroups Icterohaemorrhagiae and Canicola used for the manufacture of the leptospirosis component of vaccines are known and used in the manufacture of the polyvalent vaccine VGNKI against animal leptospirosis, a concentrated dog leptospirosis vaccine. However, we found that when combining the known strains of leptospira with new production strains of viruses in the combination according to the invention, a synergistic composition is observed, 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 Icterohaemorrhagiae (strain VGNKI-2) and serogroup Canicola (strain VGNKI-3) are contained in equal proportions with a final concentration of at least 300 million microbial cells in 1 dose of the vaccine.

Inactivation of rabies virus and leptospira is carried out in any known manner, for example, by adding formalin or ethyleneimine dimer (DEI).

As part of the proposed vaccine, suspensions of virus strains and leptospira can be contained in various combinations, which will determine the infections for the prevention of which this drug is used. In this case, suspensions may be contained in any combination by volume, however, the condition described below must be met.

The composition of 1 dose of a vaccine against plague, adenovirus infections, parvovirus and coronavirus enteritis, leptospirosis and rabies in dogs should contain at least:

carnivore plague virus 10 ^3.5 TCD ₅₀ type 2 dog adenovirus 10 ^3.0 TCD ₅₀ parvovirus dogs 10 ³ _, ⁰ GAE dog coronavirus 10 ^3.0 TCD ₅₀ rabies virus 1 ME leptospira serogroups Icterohaemorrhagiae and Canicola 3 × 10 ⁸ microbial cells

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 target additive, the 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 — aluminum 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.

If necessary, as a target additive, the proposed vaccine may contain a preservative sodium thiolate in an amount of 1: 20,000.

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 10 to 20 vol.% Adjuvant.

In a preferred embodiment, the vaccine contains 6% gel of alumina hydrate in an amount of 10 vol.%, Which corresponds to 6 g of alumina hydrate in 1 liter of vaccine.

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

The vaccine against plague, adenovirus infections, parvovirus and coronavirus enteritis, leptospirosis and dog rabies is represented by two components:

- dry (lyophilized), containing a mixture of suspensions of attenuated strains of the plague virus of carnivorous STB No. 2313, adenovirus of dogs of the second type of STB No. 2311, parvovirus of dogs of the second type of STB No. 2312 and coronavirus of dogs of STB No. 2312;

- liquid, containing inactivated suspensions of leptospira of two serogroups, inactivated suspension of the strain of the rabies virus EPA - CB-M20 and aluminum hydroxide as an adjuvant.

The dry component of the vaccine is mixed with the liquid component of the vaccine immediately before use.

The lyophilized component is a dry, homogeneous, finely porous mass of yellow-pink color. The liquid component is a homogeneous pink suspension with a precipitate at the bottom of the bottle, which, when shaken, breaks up into a homogeneous suspension.

The liquid component of the proposed vaccine is a solvent of the dry lyophilized component of the vaccine.

Before use, the dry lyophilized component of the vaccine is dissolved in the liquid component, shaken until a homogeneous suspension is obtained. When used, precipitation of the components of the vaccine is excluded. If there is a slight unbreakable sediment, the vaccine is pipetted 3-5 times in a vial using a syringe until a homogeneous suspension is obtained.

The vaccine is stable and retains its original activity at a temperature of 2-8 ° C for at least 18 months (observation period). Only clinically healthy puppies and adult dogs should be vaccinated. The vaccine is administered to puppies at 8-10 weeks of age and reapplied after 21-28 days. Revaccination of puppies is carried out at the age of 10-12 months. Adult dogs are vaccinated once a year.

The vaccine in compliance with the rules of aseptic and antiseptic is administered intramuscularly in the thigh at a dose of 2.0 cm ³ immediately after dissolution. Dogs of small and decorative breeds are vaccinated in a dose of 1 cm ³ , for which the dry component is dissolved in 1 cm ^{3 of} liquid.

The vaccine was tested on puppies and adult dogs of private owners and in dog farms. This testing confirms the safety of the drug and its effectiveness, manifested in enhancing the immunizing activity of the components of the 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 in the external environment.

In vaccinated dogs, the vaccine causes the formation of immunity to plague, adenovirus infections, parvovirus, coronavirus enteritis, leptospirosis and rabies 2–3 weeks after immunization, which lasts 6–8 months in young animals and 12–15 months in adults (observation time) .

The vaccine according to the invention is characterized by higher antigenic and immunogenic activity, the ability to create high tension colostral immunity, high storage stability.

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

Example 1. Obtaining a vaccine against plague, adenovirus infections, parvovirus and coronavirus enteritis, leptospirosis and rabies dogs.

The vaccine is prepared from new production strains of the plague virus of carnivorous STB No. 2313, adenovirus of dogs of the second type, STB No. 2311, parvovirus of dogs of the 2nd type of STB No. 2312, coronavirus of dogs STB No. 2314, rabies virus Epa - SV-M20, and known production strains of leptospira serogroups Icterohaemorrhagiae and Canicola.

The technological process consists of the following main stages:

- stages for the separate production of viral suspensions from strains of the plague virus of carnivorous bills No. 2313, adenovirus of dogs of the second type of bills No. 2311, parvovirus of dogs of the second type of bills No. 2312, coronavirus of dogs of bills No. 2314 and the rabies virus Epa - CB-M20;

- determination of the infectious activity of each viral suspension;

- the stage of separate production of bacterial cultures of leptospira serogroups Icterohaemorrhagiae and Canicola and counting the number of microbial cells per unit volume, inactivation, concentration by ultrafiltration; inactivation of virus-containing culture fluid of strain Ep - CB-M20 rabies virus;

- combining the obtained suspensions of the plague virus of carnivorous STB No. 2313, adenovirus of dogs of the second type of STB No. 2311, parvovirus of dogs of the 2nd type of STB No. 2312, coronavirus of dogs of STB No. 2314, adding a stabilizer, mixing all components of the vaccine with subsequent packaging and lyophilization; combining the obtained inactivated suspension of rabies virus and bacterial concentrated cultures of the leptospira serogroups Icterohaemorrhagiae and Canicola, adding an adjuvant, followed by packaging of the finished product.

The step of 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 a strain of the virus;

- control of the strain of the virus.

To cultivate a strain of the plague virus of carnivorous STB No. 2313, a culture of Vero cells, strains of adenovirus of dogs of the second type of STB No. 2311, parvovirus of dogs of the 2nd type of STB No. 2312, coronavirus of dogs of STB No. 2314, a culture of MDSC cells, and rabies virus Era - CB- are used. M20 - ANS cell culture.

Leptospira cultures of Leptospira interrogans serogroup Canicola (VGNKI-3 strain) of the serogroup Icterohaemorrhagiae (VGNKI-2 strain) are grown separately in liquid serum culture medium for 7-10 days before the accumulation of each leptospira strain of at least 70 million microbial cells in 1 cm ³ .

Next, the resulting culture will be combined in a 1: 1 ratio, 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.

The accumulation in the cell culture of plague viruses of carnivorous bills No. 2313, adenovirus of dogs of the second type of bills No. 2311 and coronavirus of dogs No. b. No. 2314 was evaluated by the cytopathic effect. The virus titer is calculated by the method of Reed and Mench and expressed in lg TCD ₅₀ / cm ³ .

The accumulation of parvovirus of dogs of type 2 STB No. 2312 is estimated by hemagglutinating activity using pig red blood cells and expressed in GAE / cm ³ .

The accumulation in the culture of cells of BHK-21 of the rabies virus strain SV-20M is evaluated by the volumetric method of the National Institute of Health of the USA by its immunogenic activity for mice compared to the reference strain of rabies virus and expressed in ME.

The accumulation of lepospira is determined in the Petrov-Hausser counting chamber. When the titer of the plague virus is reached, carnivorous values are 10 ^6.5 -10 ^7.0 TCD ₅₀ / cm ³ , adenovirus of dogs of the second type - 10 ^5.5 -10 ^6.5 TCD ₅₀ / cm ³ , parvovirus of dogs of the 2nd type - 10 ⁵ -4 * 10 ⁵ GAU / cm ³ , coronavirus of dogs - 10 ^5.5 -10 ^6.0 TCD ₅₀ / cm ³ , and rabies virus - 2 ME, the resulting virus-containing material of each strain is purified from ballast impurities by centrifugation or any other known method.

The combination of the obtained suspensions of the plague virus of carnivorous STB No. 2313, adenovirus of dogs of the second type of STB No. 2311, parvovirus of dogs of the 2nd type of STB No. 2312, coronavirus of dogs of STB No. 2314 is carried out in such a way that after adding stabilizer and lyophilization to 1 cm ^{3 of the} vaccine it does not contain less:

carnivore plague virus 10 ^3.5 TCD50 type 2 dog adenovirus 10 ^3.0 TCD ₅₀ parvovirus dogs 10 ^3.0 GAE dog coronavirus 10 ^3.0 TCD ₅₀ .

The result is a dry (lyophilized) component of the vaccine

The combination of the obtained inactivated suspensions of rabies virus and bacterial cultures of the leptospira serogroups Icterohaemorrhagiae and Canicola is carried out in such a way that after adding the adjuvant to 2 cm ³ (1 dose) of the vaccine, at least:

rabies virus 1 ME leptospira 3x10 microbial cells.

The result is a liquid component of the vaccine.

The dry component of the vaccine is mixed with the liquid component of the vaccine immediately before use.

Example 2. Evaluation of the antigenic activity of the vaccine against plague, adenoviral infections, parvovirus and coronavirus enteritis, leptospirosis and rabies in dogs in experimental models.

To determine the antigenic activity of the combination vaccine according to the invention, puppies of arctic foxes (dogs) aged 8-10 weeks are used as an experimental model.

In the experiment, clinically healthy puppies of Arctic foxes of both sexes, aged 8-10 weeks in the amount of 60 goals, are used. Puppies are divided into 2 groups - experimental (50 goals) and control (10 goals).

The animals of the experimental group, the vaccine is administered twice intramuscularly at a dose of 2 cm ³ with an interval of 21 days. Control puppies are not vaccinated. Prior to vaccination and after each vaccination, serum samples are taken from animals.

Paired blood serum taken from animals before and after vaccination is examined in pH for antibodies to the plague virus, dog adenovirus and coronavirus, in RTGA for antibodies to the dog parvovirus, in PMA for the presence of antibodies to leptospira and for the presence of rabies antibodies by FAVN . The experimental results are presented in table.6.

Table 6 The results of the study of the antigenic activity of the combined vaccine on Arctic fox puppies Antibody titers Geometric mean antibody titers Before introduction 21 days after the 1st vaccination 21 days after the 2nd vaccination the control experienced the control experienced the control experienced Dog Plague Virus 0 0 0 7.8 log2 0 9.4 log2 Parvovirus dogs 5.2 log2 4.4 log2 6.2 log2 8.3 log2 4.2 log2 10 log2 Dog adenovirus 0 0 0 7.9 log2 0 8.2 log2 Dog coronavirus 0 0 0 2.8 log2 0 5.5 log2 L. canicola 0 0 0 1: 200 0 1: 800 L. Icterohaemorrha-giae 0 0 0 1: 200 0 1: 1600 Rabies virus 0 0 0 1.18 IU / ml 0 7.45 IU / ml

The data in table 6 indicate that in all vaccinated animals on the 21st day after the second vaccination there was a more than four-fold increase in the titer of antibodies to all antigens that make up the vaccine. The level of anti-rabies antibodies is significantly higher than necessary (0.5 IU / ml). In control animals, seroconversion in relation to the studied antigens was not observed.

Thus, the results indicate a high antigenic and immunogenic activity of the combined vaccine against plague, adenovirus infections, parvovirus and coronavirus enteritis, leptospirosis and rabies in dogs.

Example 3. Determination of antigenic activity of the vaccine against plague, adenovirus infections, parvovirus and coronavirus enteritis, leptospirosis and rabies of dogs in relation to all components and immunogenic activity against the virus of plague of dogs.

The experiment uses clinically healthy thorzofret of both sexes, aged 10-16 weeks in the amount of 20 goals. Animals are divided into 2 groups - experimental (16 goals) and control (4 heads).

Animals of the experimental group, the vaccine is administered once intramuscularly at a dose of 2 cm ³ . Control thorzofretoks are not vaccinated. Before vaccination and on the 21st day after vaccination, blood serum samples are taken from animals.

On the 21st day after vaccination, the animals are infected with the S. hill strain of the dog plague virus. The virus is administered intramuscularly at a dose of 100 LD ₅₀ .

Analysis of the results is carried out within 14 days after infection control. Paired blood serum taken from animals before and after vaccination is examined in pH for antibodies to the plague virus, dog adenovirus and coronavirus, in RTGA for antibodies to the dog parvovirus, in PMA for the presence of antibodies to leptospira and for the presence of rabies antibodies by FAVN . The experimental results are presented in table.7.

As a result of the study, it was found that in all vaccinated animals on the 21st day after vaccination there was a more than four-fold increase in the titer of antibodies to all antigens that make up the vaccine. Clinical studies of animals conducted within two weeks after control infection showed that all vaccinated animals were resistant to infection with their virulent strain of the plague virus of dogs. Moreover, all control animals (not vaccinated) fell with a characteristic clinical picture of carnivore plague.

Table 7 The results of a study of the antigenic and immunogenic activity of a combination vaccine on thorsofret Antibody titers Geometric mean antibody titers Before introduction 21 days after vaccination the control experienced the control experienced Dog Plague Virus 0 0 0 5.75 log2 Parvovirus dogs 2.75 log2 2.25 log2 2.25 log2 7.25 log2 Dog adenovirus 0 0 0 5,251og2 Dog coronavirus 0 0 0 3.5 log2 L. canicola 0 0 0 1: 400 L.Icterohaemorrhagi ae 0 0 0 1: 200 Rabies virus 0 0 0 1.5 IU / ml Control infection results Typical clinical signs of canine distemper (rhinitis, conjunctivitis, refusal to feed), death 4-6 days after infection Animals are clinically healthy

The tests showed that the combined vaccine against plague, adenoviral infections, parvovirus and coronavirus enteritis, leptospirosis and rabies in dogs has a pronounced antigenic and immunogenic activity.

Example 4. Determination of the harmlessness of the vaccine against plague, adenoviral infections, parvovirus and coronavirus enteritis, leptospirosis and rabies dogs.

In the experiment, clinically healthy puppies of dogs aged 8-12 weeks (20 goals) and rabbits weighing 3.0-3.5 kg (20 goals) are used.

The vaccine is administered subcutaneously to the blade at a dose of 5.0 cm ³ once.

Accounting for the results of harmlessness is carried out within 21 days after administration of the drug, taking into account the clinical condition of the animals.

Clinical examinations of animals during the 21st day after injection showed that the introduction of the vaccine does not cause abnormalities in the animal's health and side effects. All animals maintained normal behavioral reactions; there was no decrease in appetite and the appearance of depression.

The results of the studies indicate the safety of the combined vaccine against plague, adenovirus infections, parvovirus and coronavirus enteritis, leptospirosis and rabies in dogs.

Example 5. Clinical trials of a vaccine against plague, adenoviral infections, parvovirus and coronavirus enteritis, leptospirosis and rabies dogs.

Twenty service dogs aged 1 to 6 years are vaccinated once. Before vaccination and three weeks after vaccination in dogs take blood. The blood serum of animals is examined in the reaction of neutralization (PH) for the presence of antibodies to the plague virus, adenoviruses and coronavirus of dogs, in the reaction of inhibition of hemagglutination (RTGA) for the presence of antibodies to parvovirus of dogs. The preventive activity of serum against leptospira is investigated on Syrian hamsters. The presence of anti-rabies antibodies in the serum is checked by immunofluorescence (FAVN). The results are shown in table.8.

The data in Table 8 indicate that vaccinated dogs showed a high level of antibodies to the plague virus, adenoviruses, and coronavirus - after a single injection of the vaccine, their neutralization (pH) reaction showed at least a twofold increase in all animals. All dogs also showed a significant increase in anti-hemagglutinin titers to dog parvovirus.

After vaccination in vaccinated dogs, the titer of rabies antibodies should be at least 0.5 MU / ml. Before vaccination, the required titer of rabies antibodies was not found in 25% of the animals, and in 15% it was 0.5 MU / ml. On day 21 after administration of the vaccine, an anti-rabies antibody level in excess of 0.5 MU / ml was observed in 100% of the dogs.

Table 8 The results of the study of paired serum samples of vaccinated dogs Dog's name Antibody titer before and after vaccination Preventive activity to Lept. (% protection) CDV (in pH) CAV (in pH) CPV (in RTGA) CCV (in pH RV (IU \ ml) BEFORE after BEFORE after BEFORE after before after BEFORE after before after but. Rex 1:16 1: 128 1:16 1: 256 1: 512 1: 2048 1: 2 1:64 1.15 1.51 10 70 but. Mukhtar 1:32 1: 128 1: 128 1: 256 1: 256 1: 2048 1: 8 1: 128 4,56 7.92 twenty 80 but. "One" 1:32 1: 128 1:32 1: 128 1: 256 1: 4096 1: 8 1: 128 0.5 4,56 twenty 80 but. "Bark" 1:16 1: 256 1:16 1: 256 1: 1024 1: 4096 1: 4 1:32 0.29 1.15 thirty 90 but. "Cinzano" 1:64 1: 512 1:32 1: 256 1: 512 1: 4096 1:16 1:32 1.14 1,61 10 90 but. "Gleb" 1:32 1: 256 1:16 1: 2048 1: 1024 1: 2048 1: 2 1: 128 0.50 1.66 thirty 60 but. Glasha 1:32 1: 128 1:16 1: 128 1: 512 1: 2048 1: 8 1:64 0.10 6.01 40 70 him. sheep "Arnica" 1:64 1: 128 <1:64 1: 128 1: 256 1: 4096 1: 8 1:32 6.01 7.92 40 80 Labrador Roy 1:16 1:32 1:32 1: 256 1: 1024 1: 2048 1: 4 :16 1.99 4.46 twenty 90 him. sheep Irma 1: 128 1: 256 1: 256 1: 512 1: 128 1: 2048 1: 4 :16 7.92 7.92 twenty one hundred him. sheep "Pleased" 1: 8 1:32 1:32 1: 128 1:32 1: 256 0 : 128 0.22 4,56 10 90 him. sheep Shera 1:32 1: 128 1:16 1:64 1: 256 > !: 4096 1: 8 : 32 7.92 7.92 40 70 him. sheep "Monster" 1: 4 1: 128 1: 8 1: 256 1: 1024 > 1: 4096 1: 4 : 64 0.22 4,56 0 70 Labrador "3ero" 1:16 1:64 1: 128 1: 256 1: 256 1: 2048 1: 4 : 32 0.84 1.46 10 60 Labrador "Siegfrey, 1:64 1: 128 1:32 1: 128 1: 512 > 1: 4096 1: 2 1:16 6.01 7.92 twenty 80 him. sheep Ike 1: 8 1:64 1:32 1:32 1: 256 1: 4096 1: 2 1: 128 6.01 7.92 twenty 90 him. sheep "Nika" 1: 4 1: 512 1: 128 :> 4096 1: 512 > 1: 4096 1: 8 1:64 4,56 6.02 10 one hundred him. sheep "Academician 1: 8 1: 128 1: 128 : 128 1: 512 1: 2048 1: 8 1:64 1.01 3.74 10 90 him. sheep "Hindu" 1: 2 1: 128 1:32 1: 128 1: 128 1: 4096 1: 4 1:32 0.17 1.32 10 80 him. sheep Arnie 1:32 1:64 1:64 1: 128 1: 1024 1: 2048 0 1:16 0.50 1.51 thirty 80

The average preventive activity of the blood serum of dogs vaccinated with the combined vaccine against plague, adenovirus infections, parvovirus and coronavirus enteritis, leptospirosis and dog rabies, against leptospira was 80%.

Example 6. Clinical trials of a combination vaccine against plague, adenoviral infections, parvovirus and coronavirus enteritis, leptospirosis and rabies dogs.

327 dogs were vaccinated at the age of 6 weeks to 5 years. Puppies and previously unvaccinated adult dogs were vaccinated twice with an interval of 21 days; previously vaccinated adult dogs - once.

Before vaccination and three weeks after vaccination in dogs take blood. Blood serum is examined in the reaction of neutralization (PH) for the presence of antibodies to the plague virus, adenoviruses and coronavirus of dogs, in the reaction of inhibition of hemagglutination (RTGA) for the presence of antibodies to dog parvovirus. The preventive activity of serum against leptospira is examined on golden hamsters. The presence of anti-rabies antibodies in the serum is checked by immunofluorescence (FAVN).

They also monitor the condition of the animals after vaccination and the presence of general and local reactions (depression, fever, soreness and swelling at the injection site, etc.).

Vaccination is considered effective if after 3 weeks in dogs there is no less than a twofold increase in antibodies to the plague virus, adenoviruses, parvovirus and coronavirus dogs; the titer of rabies antibodies is not lower than 0.5 IU / cm ³ , and the preventive activity of serum to leptospira is not lower than 40%.

The results of clinical trials are presented in Table 9, which indicate that the vaccine against plague, adenovirus infections, parvovirus and coronavirus enteritis, leptospirosis and rabies in dogs was effective in more than 99% of cases, and minor complications were observed only in 3 vaccinated animals (0, 9%).

Table 9 Clinical trials of combination vaccine Age of animals Number of animals Efficiency (%) Complications 8-16 weeks 240 98.9 In one case, swelling at the injection site In two cases, a short-term fever and lethargy Older than a year 87 99,4 Not marked Total: 327 99,2 0.9%

Example 7. Testing the safety of a vaccine against plague, adenoviral infections, parvovirus and coronavirus enteritis, leptospirosis and canine rabies for the environment.

The experiment uses clinically healthy puppies of both sexes, aged 8-10 weeks in the amount of 10 goals. Puppies are divided into 2 groups - experimental (5 goals) and control (5 goals).

Animals of the experimental group, the vaccine is administered intramuscularly at a dose of 2 cm ³ . Control puppies are not vaccinated. Control and experimental animals are kept together to identify the possibility of horizontal transmission of viral and bacterial components of the vaccine from vaccinated animals to control. Prior to vaccination and in the next 7 days, blood was taken from all animals, smears from eyes, nose, pharynx, urine, feces, where the presence of vaccine viruses was checked by PCR.

The presence of leptospira in the urine of vaccinated and contact puppies is checked by dark field microscopy and a PCR test system.

Paired blood sera from control animals were examined in pH for the presence of antibodies to the plague virus, dog adenovirus and coronavirus, in the RTGA for the presence of antibodies to the dog parvovirus, in the PMA for the presence of antibodies to leptospira and for the presence of rabies antibodies by the FAVN method. The experimental results are presented in table 10.

Table 10 The results of the detection of viral and bacterial components of the combination vaccine in vaccinated puppies Test material Release time (days after vaccination) RFP adenovirus parvovirus coronavirus leptospira Eye washings 3 3 - - - Nasal swabs 3 3 - - - Pharyngeal swabs four 3 - - - Feces - - 5 3 - Blood 2 - - - - Urine - - - - -

The results of the studies show that in all vaccinated puppies the time for the release of the vaccine plague vaccine virus into the external environment does not exceed 4 days, adenovirus and coronavirus - 3 days, parvovirus - 5 days. No leptospira was excreted by animals into the external environment. The presence of the transmission of vaccine components to contact (control) animals was not detected. In addition, contact puppies did not show seroconversion to vaccine components.

Example 8. The study of the stability of the vaccine against plague, adenoviral infections, parvovirus and coronavirus enteritis, leptospirosis and dog rabies during storage.

In the experiment, 3 series of the combined vaccine against plague, adenovirus infections, parvovirus and coronavirus enteritis, leptospirosis and rabies dogs, stored 0 (freshly prepared), 6, 9, 12 and 18 months from the date of manufacture are used.

The antigenic activity of each vaccine is determined by a single injection of 4 puppies of foxes 10-12 weeks of age in a dose of 2.0 cm ³ . Before vaccination and 21 days after the introduction of the vaccine, the blood serum is checked for the presence of specific antibodies to the plague viruses, dog adenovirus and coronavirus, and for the presence of antibodies to dog parvovirus in RTGA.

The antigenic activity of the leptospirosis component is examined in 5 rabbits weighing 3-3.5 kg, for which the vaccine is diluted with 20% hydroxal 5 times, i.e. 2 cm ^{3 of the} vaccine add 8 cm ^{3 of} 20% alumina hydrate and administered to each rabbit in compliance with aseptic rules in the ear vein once in a dose of 1.0 cm ³ . 25 days after administration of the vaccine, blood was taken from rabbits and the obtained serum was examined in PMA with leptospira strains included in the vaccine.

The immunogenicity of the anti-rabies component of vaccines is determined by the volumetric method of the US National Institute of Health on white mice.

The results of the study of the stability of the vaccine during storage are presented in tables 11, 12 and 13, which show that all vaccinated animals at the 21 day after vaccination showed more than four-fold increase in the titer of antibodies to viral antigens that are part of the vaccine.

The level of antibodies in leptospira rabbits was not lower than 1: 400. The immunogenic activity of the studied vaccine series is not lower than 1.0, which corresponds to one international unit - 1.0 IU / ml, i.e. the immunogenic activity of the vaccines in the data series is not lower than the immunogenicity of the reference sample.

Thus, the experiments indicate that the proposed vaccine against plague, adenovirus infections, parvovirus and coronavirus enteritis, leptospirosis and rabies dogs has a decisive advantage over other known vaccines in terms of the specific protection and efficacy for a susceptible group of animals, the duration of intense immunity, harmlessness for puppies and adult dogs, according to the stability of preservation of biological properties, including antigenic and immunogenic activity for 18 months (observation period). The vaccine is environmentally friendly, because causes insignificant dissemination of live viral components into the external environment, when it is impossible to transfer them horizontally to other animals and does not cause dissemination of pathogenic agents.

Table 11
Antigenic activity of the combination vaccine on
different storage periods Shelf life of the vaccine (months) Series No. Geometric mean antibody titers (log2) Before introduction After introduction Cdv Cav CPV CCV Cdv Cav CPV CCV Freshly prepared (<1 month) one 0 - 3.25 - 6.25 - 8.25 - 2 0 0 3,5 - 6.0 - 8.0 - 3 0 0 3.75 - 6.75 - 7.75 - 6 one 0 0 3.75 0 5.75 6.0 7.75 3.75 2 0 0 3,5 0 5.5 6.75 8.5 3.75 3 0 0 3.25 0 6.0 5.75 8.0 3,5 9 one 0 0 3.75 0 6.0 6.0 7.0 3.25 2 0 0 4.0 0 5.5 6.0 7.5 3.75 3 0 0 3.25 0 5.75 6.0 7.75 3.25 12 one 0 0 3.75 0 5.25 5.25 8.25 3,5 2 0 0 3.75 0 5.75 5.75 8.75 4.0 3 0 0 3,5 0 5.75 5.25 7.75 3.25 eighteen one 0 0 3.75 0 6.0 5.25 8.0 3.75 2 0 0 3.25 0 5.5 6.0 7.5 3.75 3 0 0 3.75 0 5.5 5.75 8.5 3,5

Table 12: Antigenic activity of the leptospirosis component of the combination vaccine at different storage periods. The shelf life of the vaccine (month) Series No. Antibody titer Before introduction After introduction L.can. L. ict. L.can. L. ict. Freshly prepared (<1 month) one 0 0 1: 800 1: 1600 2 0 0 1: 800 1: 800 3 0 0 1: 400 1: 400 6 one 0 0 1: 800 1: 1600 2 0 0 1: 400 1: 1600 -53 0 0 1: 1600 1: 800 9 one 0 0 1: 400 1: 400 2 0 0 1: 800 one : 1600 3 0 0 1: 400 one : 1600 12 one 0 0 1: 800 one : 1600 2 0 0 1: 800 : 800 3 0 0 1: 400 : 400 eighteen one 0 0 1: 800 : 800 2 0 0 1: 800 : 800 3 0 0 1: 400 : 400

Tab. 13. Immunogenic activity of rabies
combination vaccine component at various times
storage The shelf life of the vaccine (month) Series No. Vaccine Immunogenicity Index Freshly prepared (<1 month) one 1,5 2 1.7 3 2.0 6 one 1,5 2 1,0 3 1.7 9 one 2.0 2 1,5 3 1,0 12 one 1,5 2 1,0 3 1,5 eighteen one 1,0 2 1.7 3 1,0

Claims (4)

1. The vaccine against plague, adenovirus infections, parvovirus and coronavirus enteritis, leptospirosis and rabies dogs, containing the active substance and targeted additives, characterized in that as the active substance it contains in 1 dose of the vaccine a mixture of a suspension of an attenuated strain of the plague virus of carnivores GBV No. 2313 , family Paramyxoviridae, genus Morbillivirus with a titer of at least 10 ^3.5 TCD ₅₀ ; suspensions of an attenuated strain of adenovirus dogs of type 2 STB No. 2311, family Adenoviridae, genus Mastadenovirus with a titer of at least 10 ^3.0 TCD ₅₀ ; suspensions of an attenuated strain of parvovirus of dogs of type 2 STB No. 2312, of the family Parvoviridae, genus Parvovirus with a titer of at least 10 ^3.0 GAE; suspension of attenuated strain of coronavirus of dogs STB No. 2314, family Coronaviridae, genus Coronavirus with a titer of at least 10 ^3.0 TCD ₅₀ ; inactivated suspension of the strain of rabies virus EPA - CB-M20, family Rhabdoviridae, genus Lyssavirus in the amount of 1 ME, inactivated suspensions of leptospira serogroups Icterohaemorrhagiae and Canicola, taken in a mixture in equal proportion with the final concentration of each strain of at least 3 × 10 ⁸ inactivated microbial cells 1 dose of vaccine. 2. The vaccine according to claim 1, characterized in that, as an inactivated suspension of the leptospira serogroup Icterohaemorrhagiae, it contains an inactivated suspension of bacterial cells of the Leptospira interrogans strain VGNKI-2, and an inactivated suspension of the leptospira serogroup Canicola - an inactivated suspension of bacterial cells of the Leptospira-Krogans interrog. 3. The vaccine according to claim 1, characterized in that it contains adjuvant as a target additive. 4. The vaccine according to claim 3, characterized in that it contains aluminum hydroxide as an adjuvant.