RU2406532C1 - Method of producing associated vaccine against streptococcosis and staphylococcosis of cattle - Google Patents

Abstract

FIELD: medicine, veterinary.

SUBSTANCE: invention relates to field of veterinary medicine. Method lies in the following: carried out is selection of affected organs of dead cattle from local epizootic centre, from which suspension is prepared. After that, performed is inoculation on differential-diagnostic media and pure cultures are separated, and causative agents of streptococcosis Streptococcus bovis and staphylococcosis Staphylococcus aureus are grown separately in meat-peptone broth with addition of glucose to 0.2% concentration with titre 4-5 billion in 1 cm³. Cultures are inactivated by introduction of formalin to 0.4-0.5% final concentration and kept at temperature 37°C for 72-96 hours. After that, cultures are mixed in equal ratios, solution of aluminium hydroxide is introduced in amount 20% to the volume, thoroughly mixed and packed.

EFFECT: method is simple in application and allows to produce safe, specific, highly immunogenic vaccine.

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Description

The invention relates to veterinary medicine, in particular to a method for manufacturing vaccines, and can be used in research and production institutions involved in the design of biological products, as well as in enterprises of the biological industry.

A known method of producing an associated vaccine for the prevention of infections caused by opportunistic bacteria (RF patent for the invention No. 2035189 from 02.01.86, MKI A61K 39/125, 39/135). This method involves the separate isolation of protective antigens, the cytoplasmic antigen is isolated from the Staphylococcus aureus strain No. B - 243, the Pseudomonas aeruginosa strain NIIEM No. PA - 7 is obtained from the toxin producing strain, Pseudomonas aeruginosa toxoid, from clinical strains Proteus mirabilis No. 6, 8, 15, 15, 8, 40 4641 isolate soluble antigens by controlled proteolysis, the resulting antigens are mixed in saline based on their content in 1 ml of the vaccine 0.15-0.2 mg of protein, 15-30 μg of protein, 10-20 μg of protein, respectively, and commercial stafil coccal toxoid and aluminum hydroxide in an amount of 5-10 EU and 1-2 mg, respectively. The technology for producing the vaccine is very complex.

A known method for producing a multicomponent vaccine for immunotherapy of diseases caused by opportunistic microorganisms (RF patent for the invention No. 2083223 from 05/27/94, MKI A61K 39/116, 39/02, 39/085, 39/108). This method involves the separate cultivation of strains of Klebsiella pneumoniae GISC No. 204 on Worfel-Fergrusson medium, Proteus vulgaris GISC No. 177, Escherichia coli K-100 No. 147, followed by extraction of antigens with hydroxylamine, and from cells Staphylococcus aureus No. 5 and No. 9 and aqueous extraction antigens are mixed in a ratio of 0.6, 0.6, 0.6 and 0.8 mg, respectively, per 1 ml of distilled water. The technology for producing the vaccine is very complex.

A known method of manufacturing an associated vaccine against streptococcosis and enterococcal nutria infection (RF patent for the invention No. 2301076 from 06/20/07, MKI A61K 39/116, A61K 39/09). This method involves the selection of the affected organs from fallen nutria from the local epizootic focus, from which the suspension is prepared, inoculated on differential diagnostic media, pure cultures of pathogens are isolated, cultures of Streptococcus pneumoniae and Streptococcus faecalis are separately grown in meat-peptone broth with a titer of 4-5-5 billion microbial cells in 1 cm ³ , inactivate by adding formalin to 0.4-0.6% concentration, incubate at 37 ° C for 72-96 hours, mix the cultures in equal proportions, then add the solution OR aluminum hydroxide in an amount of 15% by volume, thoroughly mixed, packaged and sealed. This vaccine is used to prevent streptococcosis and enterococcal nutria infection; cattle are not used.

The technical solution of the problem of the invention is to remedy these shortcomings, in particular, to increase the specificity, efficiency of the method of manufacturing a vaccine against streptococcosis and staphylococcosis in cattle by introducing new pure cultures of pathogens, components, excluding negative and side effects.

The solution is achieved in that a method of manufacturing a vaccine associated against streptococcosis and staphylococcosis of cattle, including obtaining antigens, inactivation, the introduction of adjuvant, characterized in that the selection of affected organs from fallen cattle from the local epizootic focus, from which the suspension is prepared, sow on differential diagnostic media, isolate pure cultures and separately grow causative agents of streptococcosis Streptococcus bovis and staphylococcosis Staphyl ococcus aureus in meat-peptone broth with glucose added to 0.2% concentration with a titer of 4-5 billion microbial cells in 1 cm ³ , inactivated by adding formalin to 0.4-0.5% final concentration, maintained at a temperature of 37 ° C for 72-96 hours, mix the cultures in equal proportions, then add a solution of aluminum hydroxide in an amount of 20% by volume, mix thoroughly, pack and seal.

The novelty of the proposed proposal is due to the fact that, unlike the known methods, the selection of affected organs from fallen cattle during their illness and death from streptococcosis and staphylococcosis is carried out, a suspension is prepared from pathological material, pure cultures of pathogens are sown on differential diagnostic media. Used for separate cultivation of pure crops Str. bovis and staph. aureus meat-peptone broth with the addition of glucose up to 0.2% and receive a concentration of microbial cells of at least 4-5 billion in 1 cm ³ . Formalin is inactivated to a 0.4-0.6% final concentration at a temperature of 37 ° C for 72-96 hours and pure cultures are mixed in equal proportions, which allows to completely suppress pathogenicity and maintain high immunogenicity against streptococcosis and staphylococcosis. The addition of aluminum hydroxide in an amount of 20% to the culture volume allows 12-15 days after intramuscular or subcutaneous administration twice with an interval of 7-14 days in a dose of 5.0 cm ³ for cows, for calves aged 1.0-1.5 months - in a dose of 1.5-2.0 cm ³ in vaccinated animals to ensure the formation of intense immunity against two infectious diseases. The method provides a harmless, highly immunogenic, specific vaccine for protecting cattle from streptococcosis and staphylococcosis.

According to the patent and scientific and technical literature, no similar set of features has been found that 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,

Methods of isolation and characterization of streptococci and staphylococci are described in the reference book “Determinant of zoopathogenic microorganisms” edited by Professor Sidorov MA, Moscow. - Kolos, 1995, pp. 90-95; in the "Workshop on Veterinary Microbiology and Immunology" authors: Kostenko TS, Rodionova VB, Skorodumov DI, M .: Kolos, 2001, on pages 165-171; in the reference book “Microbiological diagnosis of animal bacterial diseases” authors: Skorodumov DI, Subbotin VV, Sidorov MA, Kostenko TS, M .: “IzograF”, 2005, on pages 266-257 ; in the "Guidelines for the laboratory diagnosis of animal streptococcosis", approved on September 25, 1990, by the General Directorate of Veterinary Medicine with the State Inspectorate under the State Commission of the USSR Council of Ministers for Food and Procurement.

The essence of the proposed method lies in the fact that they carry out the selection of affected organs from fallen cattle from the local epizootic focus, from which a suspension is prepared, inoculum is made on differential diagnostic media, pure cultures are isolated and cultures of Str. Streptococcosis pathogens are separately grown. bovis and staphylococcosis Staph. aureus in meat-peptone broth with the addition of glucose to 0.2% concentration with a titer of 4-5 billion microbial cells in 1 cm ³ , inactivate by adding formalin to 0.4-0.5% final concentration, incubated at temperature of 37 ° C for 72-96 hours, mix the cultures in equal proportions, then add a solution of aluminum hydroxide in an amount of 20% by volume, mix thoroughly, pack and seal.

An example of a specific implementation of the method of manufacturing a vaccine.

To produce a vaccine associated against streptococcosis and staphylococcosis in cattle, the affected organs are selected from streptococcosis and staphylococcosis that died during the period of the disease, from which a suspension is prepared and bacteriological studies are carried out in order to isolate pure cultures of pathogens.

To determine the morphology and tinctorial properties of the causative agent of staphylococcosis, fingerprints are prepared from the affected organs of the fallen animals, and they are stained using the Gram method. When microscopying the preparations under an immersion microscope system, a spherical blue shape, located by cocci groups, characteristic of the genus Staphylococcus, is observed.

To determine the cultural properties of the causative agent of staphylococcosis, cultures of the selected culture are made from pathological material into meat-peptone broth (MPB), on differential diagnostic media (salt MPA, MPB with 8-10% sodium chloride, yolk-salt agar with the addition of 0.1% crystal violet — in this medium, orange colony), on meat-peptone agar (MPA) and incubated in an incubator at 37 ° C for 18-24 hours. Smooth, transparent, colorless colonies with smooth edges grow on Petri dishes with MPA. In test tubes with BCH, intense turbidity of the nutrient medium with the formation of sediment is observed. Colonies with a smooth surface, even edges of golden color grow in Petri dishes, which is typical for the genus Staphylococcus.

Isolated pure culture of the pathogen Staphylococcosis Staph. aureus forms capsules, secretes hemolysin, fibrinolysin, gelatinase, lecithinase.

The pathogenicity and specificity of the pure isolated culture is determined by intraperitoneal infection of white mice weighing 14-18 g. After 2-4 days of observation, all infected laboratory animals die at 100% safety in the control. To confirm the specificity of the death of white mice from the parenchymal organs of fallen mice, fingerprints are prepared and stained using the Gram method. When microscoping under the immersion system of the microscope, thin red rods are observed, located singly, characteristic of Staph. aureus.

To determine the morphology and tinctorial properties of the causative agent of streptococcosis, fingerprint smears are prepared from the affected organs of the fallen nutria, and they are stained using the Gram method. Microscopy of preparations under the immersion microscope system observed small cocci in the form of chains, colored in purple, characteristic of the genus Streptococcus. To determine the cultural properties of the pathogen, crops of the isolated culture are made from the material in the meat-peptone broth with glucose up to 0.2% final concentration and 10% inactivated horse serum (MPB), in Petri dishes with meat-peptone agar (MPA) with the addition of glucose to a 0.2% final concentration and 5% defibrinated rabbit blood. After 18-20 hours of incubation in a thermostat at a temperature of 37 ° C, growth is observed in the BCH with a uniform turbidity of the medium. In Petri dishes on glucose-blood MPA, growth of round, translucent, flat, with a raised center and the edges of the colony, with an alpha-type hemolysis zone, which is typical for Str. bovis.

To differentiate streptococci from staphylococci, a catalase test is used. When staging a catalase sample, a drop of a freshly prepared 3% hydrogen peroxide solution is applied to a glass slide. Bacteriological loop remove one colony of the studied culture and rub it in a drop of hydrogen peroxide. Staphylococci, in contrast to streptococci, form catalase and cause foaming. For preliminary identification of streptococci and enterococci, the culture is seeded in test tubes with 10%, 40% bile, with 6.5% sodium chloride, with carbohydrates (raffinose, sorbitol, mannitol), growth in a liquid medium and hemolysis on MPA with blood are taken into account.

As a result, diffuse growth of baculture is observed in test tubes with bile, fermentation of mannitol is noted. On blood MPA, incomplete hemolysis of red blood cells is observed with the formation of a greenish zone around the colonies, which is typical for streptococci.

To establish the serogroup affiliation of streptococci, streptococcal group precipitating serums are used. The serogroup affiliation of streptococci is determined in the precipitation reaction (RP) in the capillaries. Pathogenicity is confirmed by bioassay on young white mice.

Pure cultures of Staph pathogens Staph. aureus and streptococcosis Str. bovis is used to obtain the vaccine.

The cultivation of pure cultures is carried out separately in meat-peptone broth. For infection take 5 cm ³ fresh clean culture of the pathogen Staphylococcosis Staph. aureus per 1000 cm ³ meat-peptone culture medium and grown at 37 ° C for 18-24 hours. The concentration of microbial cells in the bacterial mass is determined according to the turbidity standard and diluted with sterile distilled water to a concentration of 4-5 billion in 1 cm ³ . The inactivation of the obtained raw materials is carried out by introducing formalin of 0.4-0.5% final concentration, kept at a temperature of 37 ° C for 72-96 hours with periodic stirring.

For infection take 5 cm ³ pure culture of the causative agent of streptococcosis Str. bovis per 1000 cm ^{3 of} liquid nutrient medium with the addition of 0.2% glucose and grown at 37 ° C for 12-14 hours. The concentration of microbial cells in the bacterial mass is determined according to the standard and diluted with sterile distilled water to a concentration of 4-5 billion in 1 cm ³ . Inactivation of bakery is carried out separately by adding formalin of 0.4-0.5% final concentration, kept at a temperature of 37 ° C for 72-96 hours with periodic stirring, inactivated baculture is mixed in equal proportions. Then, a solution of aluminum hydroxide in an amount of 20% by volume is added to the baculture mixture and thoroughly mixed, packaged and corked.

Thus, the use for the manufacture of a vaccine associated with streptococcosis and staphylococcosis in cattle pure cultures of causative agents of streptococcosis Str. bovis and staphylococcosis Staph. aureus, isolated from a local epizootic focus, provides a specific, highly immunogenic associated vaccine to protect against these two infectious diseases: streptococcosis and staphylococcosis of cattle aureus Staphylococcus aureus. Separate growth allows to obtain a concentration of bacterial cells of at least 4 billion in 1 cm ³ during 12-14 hours of incubation. The use of formalin for inactivation in a 0.4-0.5% final concentration makes it possible to suppress pathogenicity for 72-96 hours at a temperature of 37 ° C and maintain high immunogenicity for 12 months of storage. Adsorption of two antigens on aluminum hydroxide allows 12-15 days after intramuscular or subcutaneous administration twice with an interval of 7-14 days in a dose of cows of 5.0 cm ³ , calves aged 1.0-1.5 months in a dose of 1, 5-2.0 cm ³ in vaccinated animals to ensure the formation of intense immunity against two infectious diseases lasting at least 12 months (table).

The proposed method is simple to implement, affordable and is industrially applicable.

Comparative characteristics of the manufacture of the vaccine according to the proposed method and prototype No. p / p Features Prototype The proposed method one. Pathogens Strains of Streptococcus pneumoniae and Streptococcus faecalis isolated from a local epizootic focus Pure baculture of causative agents of streptococcosis Streptococcus bovis and staphylococcosis Staphylococcus aureus isolated from the local epizootic focus 2. Isolation of soluble antigens All full antigens are used. All full antigens are used. 3. Inactivation of the bacterial mass 0.4-0.5% formalin concentration for 72-96 hours at 37 ° C 0.4-0.5% formalin concentration for 72-96 hours at 37 ° C four. Components aluminum hydroxide 15% by volume aluminum hydroxide 20% by volume 5. Immunity duration 9 months 12 months

Thus, these tables show the advantages of the proposed method for the manufacture of a vaccine associated against streptococcosis and staphylococcosis of cattle in comparison with the existing prototype.

Claims (1)

A method of manufacturing a vaccine associated against streptococcosis and staphylococcosis of cattle, including the production of antigens, inactivation, the introduction of an adjuvant, characterized in that they select the affected organs from the fallen cattle from the local epizootic focus, from which the suspension is prepared, and the differential diagnostic test is inoculated media, pure cultures are isolated and the causative agents of streptococcosis Streptococcus bovis and staphylococcosis Staphylococcus aureus are separately grown in meat-peptone broth with the addition of glucose to 0.2% concentration with a titer of 4-5 billion microbial cells in 1 cm ³ , inactivate by adding formalin to 0.4-0.5% final concentration, incubated at a temperature of 37 ° C for 72 -96 h, mix the cultures in equal proportions, then add a solution of aluminum hydroxide in an amount of 20% by volume, mix thoroughly, pack and seal.