RU2510839C2 - Associated salmonellosis, escherichiosis and rabbit viral haemorrhagic disease vaccine - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to veterinary medicine, and concerns an associated salmonellosis, escherichiosis and rabbit viral haemorrhagic disease vaccine. The presented vaccine contains antigens in the form of pure-growth suspended cells of salmonellosis Sal.typhimurium, escherichiosis E.coli and rabbit viral haemorrhagic disease prepared from target affected organ of dead rabbits in a local epizootic centre; the vaccine also contains glucose, formalin and aluminium hydroxide in the specified amounts.

EFFECT: presented invention enables providing higher specificity and efficacy of the prepared vaccine, as well as eliminating side effects in the animals and may be used for biopharmaceutical engineering.

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Description

The invention relates to veterinary medicine, in particular to associated vaccines, and can be used in institutions involved in the construction of biological products, as well as in enterprises of the biological industry.

Known associated vaccine against calf diarrhea containing bacterial cells of Escherichia coli strains: 0138 K88,09K99, 200; genus Salmonella, strains: GISC No. 195, GISC No. 196, GISC No. 197; r0 Yes Proteus, strains: GISK No. 198, GISK No. 199; Klebsiella pneumoniae, strains: GISK No. 201, GISK No. 202 (patent for the invention of the Russian Federation No. 2035916 dated 06/17/93, MKI A61 39/125, 39/135). This vaccine is intended for the prevention of infectious diseases in calves caused by Escherichia, Salmonella, Protein and Klebsiella.

Known inactivated emulsion vaccine against calf salmonellosis (RF patent for the invention No. 2154495 from 10.15.97, MKI A61K 39/112, C12N 1/20, A61K 39/39). This vaccine contains, as antigens, a mixture of formalin-inactivated virulent strains of the genus Salmonella (S. dublin No. 1449, S. typhimurium No. 159) as an immunomodulator, a suspension of Rodococcus equi No. 2 cells in an oil-lanolin mixture. This vaccine is used to prevent calf salmonellosis. The use of the oil-lanolin mixture in the vaccine can cause various vaccine complications in vaccinated animals (inflammatory processes, abscesses at the injection site, lameness, etc.)

The technical result of the invention is to obtain a harmless, highly immunogenic, storage stable vaccine against salmonellosis, escherichiosis and viral hemorrhagic rabbit disease.

The technical result is achieved in that in a vaccine associated against salmonellosis, escherichiosis and viral hemorrhagic rabbit disease containing a suspension of inactivated antigens of S. typhimurium, E. coli and rabbit viral hemorrhagic disease, the adjuvant according to the invention contains cell suspension of pure cultures of salmonella pathogens as antigens S. typhimurium, E. coli Escherichiosis and rabbit hemorrhagic disease virus in equal proportions obtained by selection of affected organs from dead rabbits from local epizootic focus, as well as formalin and aluminum hydroxide, while preparing a suspension of S. typhimurium and E. Coli antigens by preparing a suspension of affected organs from dead rabbits, plating on differential diagnostic media, growing separately in meat-peptone broth with glucose up to the concentration of microbial cells 4-5 billion in 1 cm ³ and the selection of pure cultures of pathogens of salmonellosis S. typhimurium, Escherichiosis. Coli, and a suspension of cells of a pure culture of the causative agent of the rabbit hemorrhagic disease virus virus isolated from the affected organ of the fallen rabbit from the local epizootic focus is obtained after infection with the indicated rabbit virus in the form of a 10-15% suspension of liver cells from infected rabbits, with virus activity of at least 10 ^3.0 LD ₅₀ / cm ^{3, the} obtained cell suspension were mixed pure culture of the pathogen salmonella S. typhimurium, a pure culture of cells of the pathogen E. coli ehsherihioza suspension of liver cells and rabbit haemorrhagic virus Usable rabbits in the following ratio, wt.%:

cell suspension of a pure culture of the pathogen of salmonellosis S. typhimurium isolated from a local epizootic focus in a nutrient medium with a titer of 4-5 billion microbial cells in 1 cm ³ 24.0-29.0 cell suspension of a pure culture of the pathogen Escherichiosis E. coli isolated from a local epizootic focus in nutrient medium with a titer of 4-5 billion microbial cells in 1 cm ³ 24.0-29.0 rabbit liver cell suspension with hemorrhagic virus rabbit disease isolated from local epizootic foci in physiological saline with activity of at least 10 ^3.0 LD ₅₀ / cm ³ 24.0-29.0 glucose 2.0-1.0 formalin 2.0-1.5 aluminum hydroxide rest

The novelty of the claimed technical solution is due to the fact that, in contrast to the known associated vaccines, the affected organs are selected from dead rabbits from the local epizootic focus, suspension is prepared, culture on differential diagnostic media, isolation of pure cultures of S. typhimurium salmonellosis pathogens, Escherichiosis E coli and the virus of viral hemorrhagic disease of rabbits, the cultivation of pure cultures of pathogens is carried out separately, which allows to increase the specificity and immunogenicity in ktsiny.

Separate cultivation of pure cultures of pathogens gives a concentration of S. typhimurium and E. coli of at least 4-5 billion in 1 ml for 12-14 hours of incubation and the virus of viral hemorrhagic disease of rabbits with an activity of at least 10 ^3.0 LD ₅₀ / cm ³ . Inactivation of bakerya with formalin in a 0.1-0.4% final concentration at a temperature of 37 ° C for 72-96 hours allows to suppress the pathogenicity of pathogens and preserve their immunogenic properties for 12 months. Mixing inactivated antigens of S. typhimurium, E. coli and rabbit hemorrhagic disease virus in equal proportions, the addition of aluminum hydroxide and thorough mixing does not cause post-vaccination complications in vaccinated rabbits, it allows ensuring the formation of intense immunity 12-15 days after two administration with an interval of 7- 10 days in a dose of 1.5 cm ³ for rabbits against salmonellosis, Escherichiosis and rabbit viral hemorrhagic disease, lasting at least 12 months. The use of pure cultures of S. typhimurium salmonellosis pathogens, E. coli Escherichiosis, and rabbit hemorrhagic disease virus isolated from the local epizootic focus from sick and dead animals can significantly increase the specificity of the vaccine, after vaccination it protects animals immediately against three dangerous infectious diseases of salmonellosis, Escherichiosis and viral hemorrhagic disease of rabbits.

According to the patent and scientific and technical literature, no similar combination of features, components was found, which allows us to judge the inventive step of the claimed technical solution.

Regarding the criterion of "industrial applicability", the components that make up the vaccine are known and widely used in veterinary practice in the manufacture of inactivated vaccines: the associated vaccine against salmonellosis, pasteurellosis and enterococcal infection of piglets TU 10.09-62-90, approved 01.08.90 g ., instructions for the manufacture and control of this vaccine approved by the General Directorate of Veterinary Medicine 07/16/90 vaccine against enterococcal infection of calves, lambs and piglets TU 10.09-91-90, approved 15.12.90, provides all the necessary information about microorganisms. However, for rabbits, these vaccines are not used.

Methods of isolation and characterization of salmonella are described in the guidelines "Laboratory diagnosis of salmonella in humans and animals, the detection of salmonella in feed, food and environmental objects." Moscow, VO "Agropromizdat", 1990, as well as in the directory "Key to Zoopathogenic Microorganisms" edited by Professor Sidorov M.A. Moscow: Kolos, 1995, pp. 201-204. In the "Workshop on Veterinary Microbiology and Immunology" authors: Kostenko TS, Rodionova VB, Skorodumov DI M .: Kolos, 2001, on pages 222-233.

The methods for isolating the pure culture of the causative agent of Escherichiosis and the characteristics are described in the “Methodological guidelines for the bacteriological diagnosis of colibacteriosis (Escherichiosis) of animals” approved on July 27, 2000 No. 13-7-2 / 2117 by the Department of Veterinary Medicine of the Ministry of Agriculture of the Russian Federation. zoopathogenic microorganisms ”edited by Professor Sidorov MA, Moscow: Kolos, 1995, pp. 169-201, as well as in the“ Workshop on Veterinary Microbiology and Immunology ”authors: Kostenko TS, Rodionova VB, Skorodumov D.I., M .: "Ear", 2001, and str.219-222.

Methods of isolation and characterization of rabbit viral hemorrhagic disease are described in the "Guidelines for the laboratory diagnosis of rabbit viral hemorrhagic disease", approved by the State Administration for Agriculture of the USSR on 12.28.88. g .; in the book "Viral hemorrhagic disease of rabbits" authors: Shevchenko AA, Vishnyakov I.F., Bakulov I.A., Vlasova T.A. M .: “Two Crowns”, 1995, 48 p .; in the book “Rabbit Diseases”, authors: Shevchenko A.A., Shevchenko L.V., Litvinov A.M. M .: "Aquarium", 2002, p. 42-57.

Associated vaccine against salmonellosis, escherichiosis and viral hemorrhagic rabbit disease, containing cell suspensions of formalin-inactivated Salmonellosis pathogens S. typhimurium, E. coli Escherichiosis and rabbit hemorrhagic disease virus adsorbed on alumina hydrate. In appearance, the vaccine is a light yellow liquid with a loose precipitate, which, when shaken, easily breaks into a homogeneous suspension. Shelf life in a dry, dark place at a temperature of 2 to 10 ° C for 1 year. The vaccine is intended for the prevention of dangerous infectious diseases against salmonellosis, Escherichiosis and viral hemorrhagic disease of rabbits. It causes the formation of specific immunity against salmonellosis, Escherichiosis and viral hemorrhagic disease of rabbits. The vaccine is harmless and areactogenic. Immunity in rabbits vaccinated with the vaccine against salmonellosis, escherichiosis and viral hemorrhagic rabbit disease, is formed 12-15 days after double administration with an interval of 7-10 days in a dose of 1.0 cm ³ against salmonellosis, Escherichiosis and viral hemorrhagic rabbit disease, lasting not less than 12 months. The vaccine is used in farms in dysfunctional and threatened by salmonellosis, escherichiosis and viral hemorrhagic disease of rabbits in the doses indicated above. Slaughter products of vaccinated animals are used without restriction, regardless of the timing of vaccination.

The factors that determine the receipt of a vaccine of a given property include the sequence of manufacture and the percentage ratio between the components included in the preparation.

Five containing components in various proportions per 100 ml of vaccine are given.

Example 1

The affected organs are taken from the dead rabbits in the epizootic focus, from which the suspension is prepared, inoculated on differential diagnostic media, pure cultures of S. typhimurium salmonellosis pathogens, E. coli Escherichiosis and rabbit hemorrhagic disease virus are isolated, separately cultivated, obtained, inactivated and mixed (wt.%) a suspension of microbial cells 66.0, with a titer of 4-5 billion, and a glucose content of 0.5, inactivate by adding formalin 1.0, then add aluminum hydroxide - the rest, mix thoroughly and get a vaccine Inu of the following composition in the following ratio of components, wt.%:

cell suspension of a pure culture of the pathogen of salmonellosis S. typhimurium isolated from a local epizootic focus in a nutrient medium with a titer of 4-5 billion microbial cells in 1 cm ³ 22.0 cell suspension of a pure culture of the pathogen Escherichiosis E. coli isolated from a local epizootic focus in nutrient medium with a titer of 4-5 billion microbial cells in 1 cm ³ 22.0 rabbit liver cell suspension with hemorrhagic virus rabbit disease isolated from local epizootic foci in physiological saline with activity of at least 10 ^3.0 LD ₅₀ / cm ³ 22.0 glucose 0.5 formalin 1,0 aluminum hydroxide rest

With this ratio of components, the vaccine associated against salmonellosis, escherichiosis and viral hemorrhagic rabbit disease has immunogenic activity.

Example 2

The affected organs are taken from the dead rabbits in the epizootic focus, from which the suspension is prepared, inoculated on differential diagnostic media, pure cultures of S. typhimurium salmonellosis pathogens, E. coli Escherichiosis and rabbit hemorrhagic disease virus are isolated, separately cultivated, obtained, inactivated and mixed (wt.%) a suspension of microbial cells 72.0, with a titer of 4-5 billion, and a glucose content of 1.0, inactivate by adding formalin 1.5, then add aluminum hydroxide - the rest, mix thoroughly and get vaccine Inu of the following composition in the following ratio of components, wt.%:

cell suspension of a pure culture of the pathogen of salmonellosis S. typhimurium isolated from a local epizootic focus in a nutrient medium with a titer of 4-5 billion microbial cells in 1 cm ³ 24.0 cell suspension of a pure culture of the pathogen Escherichiosis E. coli isolated from a local epizootic focus in nutrient medium with a titer of 4-5 billion microbial cells in 1 cm ³ 24.0 rabbit liver cell suspension with hemorrhagic virus rabbit disease isolated from local epizootic foci in physiological saline with activity of at least 10 ^3.0 LD ₅₀ / cm ³ 24.0 glucose 1,0 formalin 1,5 aluminum hydroxide rest

With this ratio of components, the vaccine associated with salmonellosis, escherichiosis and viral hemorrhagic rabbit disease has moderate immunogenic activity.

Example 3

The affected organs are taken from the dead rabbits in the epizootic focus, from which the suspension is prepared, inoculated on differential diagnostic media, pure cultures of S. typhimurium salmonellosis pathogens, E. coli Escherichiosis and rabbit hemorrhagic disease virus are isolated, separately cultivated, obtained, inactivated and mixed (wt.%) a suspension of microbial cells 84.0, with a titer of 4-5 billion, and a glucose content of 1.7, inactivate by adding formalin 1.3, then add aluminum hydroxide - the rest, mix thoroughly and get vaccine Inu of the following composition in the following ratio of components, wt.%:

cell suspension of a pure culture of the pathogen of salmonellosis S. typhimurium isolated from a local epizootic focus in a nutrient medium with a titer of 4-5 billion microbial cells in 1 cm ³ 28.0 cell suspension of a pure culture of the pathogen Escherichiosis E. coli isolated from a local epizootic focus in nutrient medium with a titer of 4-5 billion microbial cells in 1 cm ³ 28.0 rabbit liver cell suspension with hemorrhagic virus rabbit disease isolated from local epizootic foci in physiological saline with activity of at least 10 ^3.0 LD ₅₀ / cm ³ 28.0 glucose 1.7 formalin 1.3 aluminum hydroxide rest

With this ratio of components, the vaccine associated against salmonellosis, escherichiosis and viral hemorrhagic rabbit disease has immunogenic activity.

Example 4

The affected organs are taken from the dead rabbits in the epizootic focus, from which the suspension is prepared, inoculated on differential diagnostic media, pure cultures of S. typhimurium salmonellosis pathogens, E. coli Escherichiosis and rabbit hemorrhagic disease virus are isolated, separately cultivated, obtained, inactivated and mixed (wt.%) a suspension of microbial cells 87.0, with a titer of 4-5 billion, and a glucose content of 1.0, inactivated by the introduction of formalin 1.5, then add aluminum hydroxide - the rest, mix thoroughly and get vaccine Inu of the following composition in the following ratio of components, wt.%:

cell suspension of a pure culture of the pathogen of salmonellosis S. typhimurium isolated from a local epizootic focus in a nutrient medium with a titer of 4-5 billion microbial cells in 1 cm ³ 29.0 cell suspension of a pure culture of the pathogen Escherichiosis E. coli isolated from local epizootic foci in a nutrient medium with a titer of 4-5 billion microbial cells 1 cm ³ 29.0 rabbit liver cell suspension with hemorrhagic virus rabbit disease isolated from local epizootic foci in physiological saline with activity of at least 10 ^3.0 LD ₅₀ / cm ³ 29.0 glucose 1,0 formalin 1,5 aluminum hydroxide rest

At this ratio of components, the vaccine associated with salmonellosis, escherichiosis and viral hemorrhagic disease of rabbits has significant immunogenic activity, forms pronounced intense immunity after vaccination, does not cause post-vaccination complications, and is stable during storage for 12 months (table 1).

Example 5

The affected organs are taken from the dead rabbits in the epizootic focus, from which the suspension is prepared, inoculated on differential diagnostic media, pure cultures of S. typhimurium salmonellosis pathogens, E. coli Escherichiosis and rabbit hemorrhagic disease virus are isolated, separately cultivated, obtained, inactivated and mixed (wt.%) a suspension of microbial cells of 90.0, with a titer of 4-5 billion, and a glucose content of 2.0, inactivated by the introduction of formalin 1.5, then add aluminum hydroxide - the rest, mix thoroughly and get vaccine Inu of the following composition in the following ratio of components, wt.%:

cell suspension of a pure culture of the pathogen of salmonellosis S. typhimurium isolated from a local epizootic focus in a nutrient medium with a titer of 4-5 billion microbial cells in 1 ^cm3 30,0 cell suspension of a pure culture of the pathogen Escherichiosis E. coli isolated from local epizootic foci in a nutrient medium with a titer of 4-5 billion microbial cells in 1 cm ³ 30,0 rabbit liver cell suspension with hemorrhagic virus rabbit disease isolated from local epizootic foci in physiological saline with activity of at least 10 ^3.0 LD ₅₀ / cm ³ 30,0 glucose 2.0 formalin 1,5 aluminum hydroxide rest

With this ratio of components, the vaccine associated with salmonellosis, escherichiosis and viral hemorrhagic disease of rabbits has immunogenic activity, forms intense immunity, and in some animals, administration of the drug causes mild irritation.

Table Comparative vaccine efficacy Vaccine Vaccine dose
cm ³ Method of administration Immunobiological properties The number of rabbits in the experiment (goal) The presence of post-vaccination of complications Duration of immunity (months) Protection, (%) According to the prototype 1,0 Intramuscularly 1000 no 9 80-90 According to the proposed
way 1,0 Intramuscularly 1000 no 12 90-100

The table shows that the proposed vaccine associated against salmonellosis, escherichiosis and viral hemorrhagic rabbit disease has significant advantages compared with the vaccine of the prototype.

The proposed vaccine associated against salmonellosis, Escherichiosis and viral hemorrhagic disease of rabbits is simple to implement, affordable and is industrially applicable.

Claims (1)

Associated vaccine against salmonellosis, escherichiosis and viral hemorrhagic disease of rabbits, containing a suspension of inactivated antigens of S. typhimurium, E. coli and viral hemorrhagic disease of rabbits, adjuvant, characterized in that as antigens contains a suspension of cells of pure cultures of S. typhimurium salmonellosis pathogens, E. coli and rabbit hemorrhagic disease virus in equal proportions obtained by selection of affected organs from dead rabbits from a local epizootic focus, as well as formalin and aluminum hydroxide, while preparing a suspension of S. typhimurium and E. coli antigens by preparing a suspension of affected organs from dead rabbits, plating on differential diagnostic media, growing separately in meat-peptone broth with glucose to a concentration of microbial cells of 4-5 billion. 1 cm ³ and the isolation of pure cultures of Salmonella pathogens S.typhimurium, ehsherihioza E. coli, and a cell suspension of a pure culture of the pathogen rabbit haemorrhagic disease virus isolated from affected organ fallen from rabbit topically of epizootic outbreak, obtained after infection of said virus in rabbits as a 10-15% suspension of liver cells from infected rabbits, with an activity of at least 10 ^3.0 LD ₅₀ / cm ^{3, the} obtained mixed suspension of a pure culture of the pathogen Salmonella cells S.typhimurium cells of a pure culture of the causative agent of E. coli Escherichiosis and a suspension of rabbit liver cells with rabbit hemorrhagic disease virus in the following ratio of components, wt.%:
cell suspension of a pure culture of the pathogen of salmonellosis S.typhimurium isolated from local epizootic foci in a nutrient medium with a titer of 4-5 billion microbial cells in 1 ^cm3 24.0-29.0 cell suspension of a pure culture of the causative agent of Escherichiosis E. coli isolated from a local epizootic focus in nutrient medium with a titer of 4-5 billion microbial cells in 1 cm ³ 24.0-29.0 rabbit liver cell suspension with hemorrhagic virus rabbit disease isolated from local epizootic foci in physiological saline with activity of at least 10 ^3.0 LD ₅₀ / cm ³ 24.0-29.0 glucose 2.0-1.0 formalin 2.0-1.5 aluminum hydroxide rest