RU2707289C1 - Method for producing vaccine associated with colibacillosis, streptococcus and pseudomonosis of bovine animals - Google Patents

Abstract

FIELD: medicine; veterinary.

SUBSTANCE: invention refers to veterinary medicine, namely to a method for producing a vaccine associated with colibacillosis, streptococcus and pseudomonosis of cattle. Method involves selection of affected organs from diseased and dead animals from local epizootic focus, from which a suspension is prepared and seeding on differential diagnostic media, after which pure cultures of pathogens are isolated. Separately, E. coli, Str. galloliticus and Ps. aeruginosa are grown in meat-and-peptone broth with addition of glucose to 0.2 % concentration with titre of 4–5 billion microbial cells per 1 cm³. Thereafter, cultures of the disease pathogens are inactivated with formalin to a final concentration of not more than 0.4 % final concentration at temperature of 37 °C for 72–96 hours. Adjuvant is then added to the mixture of inactivated crops, in the form of an aluminium hydroxide gel in amount of 20 % by volume, before the end product is thoroughly mixed.

EFFECT: present method enables producing a vaccine associated with cattle stable in storage, associated with colibacillosis, streptococcus and cattle pseudomonosis.

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Description

The invention relates to the field of veterinary medicine, in particular to microbiology and can be used in research and production institutions involved in the development and construction of vaccines, as well as in enterprises of the biological industry.

A known analogue method of manufacturing a vaccine associated against escherichiosis, streptococcosis and staphylococcosis in cattle (RF patent for the invention No. 2538158 dated 12/30/2013, MKI A61 / K 39/116, 39/108), including the separate production of antigens with their subsequent mixing, introduction of an adjuvant, according to the invention, pathological material is taken from the sick and fallen cattle from the local epizootic focus, then a suspension is prepared from the selected pathological material and then the diffusion is inoculated potential diagnostic medium, after which cells of pure cultures of Escherichia coli Escherichia coli, Streptococcus pneumoniae streptococcosis and Staphylococcus aureus staphylococcosis pathogens are isolated, then pure culture cells of Escherichiosis pathogens, streptococcosis and glucose staphylococcosis are added separately to meat - 0.2% concentration with a titer of 4-5 billion microbial cells in 1 cm ³ , then cells of pure cultures of causative agents of Escherichiosis, streptococcosis and staphylococcosis with formalin are separately inactivated to 0.4-0.5% of the final concentration at a temperature of 37 ° C for 72-96 hours with periodic stirring, then cells of pure cultures of Escherichia coli, Escherichia coli, streptococcosis Streptococcus pneumoniae and Staphylococcus aureus staphylococcosis in equal proportions are mixed, then the adjuvant is used in the mixture of inactivated cells of pure cultures, which is used as an adjuvant aluminum hydroxide in an amount of 20% by volume, and before packing, the final product is thoroughly mixed.

The technical result of the invention is to obtain a harmless, specific, highly immunogenic associated vaccine for the protection of cattle against Escherichiosis, streptococcosis and pseudomonosis by introducing new pathogens, components, eliminating the negative and side effects.

A method of manufacturing a vaccine associated against Escherichiosis, streptococcosis and pseudonomy in cattle, comprising obtaining antigens in the form of suspensions prepared from samples of pathological material taken from a sick and fallen cattle from a local epizootic focus, plating on differential diagnostic media, isolation of pure culture cells pathogens of Escherichia coli Escherichiosis, Streptococcus streptococcosis, with their separate cultivation in meat-peptone broth with the addition of glucose up to 0.2% concentration uu 4-5 billion a titer of microbial cells per 1 cm ^3, the separate inactivation of pure cultures of cells ehsherihioza pathogens Escherichia coli, Streptococcus streptococcosis 0.4-0.5% formalin to a final concentration of strength at a temperature of 37 ° C for 72-96 hours with periodic stirring, mixing them in equal proportions, introducing into the mixture of inactivated cells pure cultures of adjuvant, which is used aluminum hydroxide in an amount of 20% by volume, thorough mixing and repeated thorough mixing, before packing the final product from characterized by the fact that cells of a pure culture of pathogens of pseudomonosis Pseudomonas aeruginosa and streptococcosis Streptococcus gallolyicus are additionally isolated, which are separately grown in meat-peptone broth with glucose added to 0.2% concentration with a titer of 4-5 billion microbial cells in 1 cm ³ , they are separately inactivated by formalin to a 0.4-0.5% final concentration at 37 ° C for 72-96 hours with periodic stirring and cells of pure cultures of Escherichia coli, Escherichia coli, pseudomonas Pseudomonas aeruginosa and streptococcus pathogens are mixed and Streptococcus gallolyicus in equal proportions.

The novelty of the proposed proposal is due to the fact that, in contrast to the known methods, pathological material is selected from a sick and fallen cattle from a local epizootic in case of Escherichiosis, streptococcosis and pseudomonosis, from which a suspension is prepared, cells of pure cultures of pathogens are sown on differential diagnostic media , cells of pure cultures of Escherichia coli Escherichiosis causative agents, Streptococcus pneumoniae streptococcosis and Pseudomonas aeruginosa pseudomon are separately grown and a concentration is obtained microbial cells of at least 4-5 billion in 1 cm3, inactivate formalin and mix inactivated cells of pure cultures in equal proportions, which allows to completely suppress pathogenicity and maintain high immunogenicity against escherichiosis, streptococcosis and pseudomonosis.

According to the patent and scientific and technical literature, no similar set of features has been found that allows judging the inventive step of the claimed technical solution.

As for the criterion of “industrial applicability”, the components included in the vaccine composition are known and widely used in veterinary practice in the manufacture of inactivated vaccines: instructions for the manufacture and control of polyvalent aluminum hydroxide aluminum formol vaccine against colibacteriosis (Escherichiosis) of piglets, calves and lambs, approved by the GUV Ministry of Agriculture of the USSR on 26.04 .1984; associated vaccine against salmonellosis, pasteurellosis and enterococcal infection of piglets TU 10.09-62-90, approved 01.08.90, instructions for the manufacture and control of this vaccine approved by the General Directorate of Veterinary Medicine on July 16, 90 provide all the necessary information about microorganisms.

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, describes the characteristics of Escherichia in the guide "Key of zoopathogenic microorganisms ”edited by Professor Sidorov MA, Moscow-Kolos, 1995, pp. 196-201, as well as in the“ Workshop on Veterinary Microbiology and Immunology ”authors: Kostenko TS, Rodionova VB, Skorodumov D.I., M .: "Ear", 2001, on pages 219-222.

Methods of isolation and characterization of streptococci are presented in the Methodological Instructions 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, and also in the reference book “Identifier of zoopathogenic microorganisms” Edited by Professor Sidorov M.A., Moscow. - Kolos, 1995, pp. 90-95; in the reference book “Microbiological diagnosis of animal bacterial diseases” authors: Skorodumov DI, Subbotin VV, Sidorov MA, Kostenko TS, M .: “IzograF”, 2005, on pages 249-257 .

Methods of isolation and characterization of microorganisms of the genus Pseudomonas are described in the "Workshop on Veterinary Microbiology and Immunology" authors: Kostenko TS, Rodionova VB, Skorodumov DI, M .: Kolos, 2001, p. 259 -261; in the reference book “Microbiological diagnosis of animal bacterial diseases” authors: Skorodumov DI, Subbotin VV, Sidorov MA, Kostenko TS, M .: “IzograF”, 2005, on pages 522-525 .

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

For the manufacture of a vaccine associated against Escherichiosis, streptococcosis and pseudomonosis in cattle, the selection of affected organs from sick and dead animals during the period of their illness with Escherichiosis, streptococcosis and pseudomonosis is carried out, from which a suspension is prepared and laboratory studies are performed to isolate pure cultures of pathogens.

To determine the morphology and tinctorial properties of the causative agent of Escherichiosis, smears are prepared - fingerprints from the affected organs of the fallen nutria, by touching fat-free glass to a fresh section of the affected tissue and subsequent fixation over the flame of an alcohol lamp, they are stained using the Gram method. When microscopying the preparations under the immersion system of the microscope, thick sticks with rounded red ends, located singly, are observed, which is typical for E. coli.

To determine the cultural properties of the causative agent of Escherichiosis E. coli, the selected culture is inoculated from pathological material into meat - peptone broth (MPB), on Endo medium, on meat - peptone agar (MPA) and incubated in an incubator at 37 ° C for 16-24 hours. Wet colonies with smooth edges and a smooth gray surface grow on Petri dishes with MPA. In test tubes with BCH, a uniform turbidity of the nutrient medium was observed with a small precipitate breaking upon shaking. In Petri dishes on Endo medium, raspberry-red colonies with pink rims 2-3 mm in diameter, characteristic of E. coli, are observed.

When determining the biochemical activity of pure bacultures, crops are sown on differential diagnostic media with a different set of components. E. coli is characterized by the breakdown of glucose and lactose with the formation of acid and gas, the release of indole, and the absence of urease.

The pathogenicity of pure isolated cultures 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 mice from the parenchymal organs of fallen mice, smears — fingerprints — are prepared and stained using the Gram method. When microscoping under the immersion system of the microscope, thick sticks with rounded red ends, located singly, characteristic of E. coli, are visible in the preparations. Serological typification of Escherichia coli is carried out in an agglutination reaction with a set of type-specific sera according to the standard technique in test tubes.

In the agglutination reaction, the test culture of Escherichia coli reacts with colic serum and a flocculent precipitate (positive result) is visible with a negative control.

To determine the morphology and tinctorial properties of the causative agent of streptococcosis, smears are prepared - fingerprints from the affected organs of the fallen nutria, by touching fat-free glass to a fresh section of the affected tissue and subsequent fixation over the flame of an alcohol lamp, 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 by adding glucose to 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 in the form of diffuse turbidity of the medium is observed in the BCH. In petri dishes with blood MPA, growth of small dewy, slightly turbid with flat edges of colonies surrounded by a greenish hemolysis zone is observed, which is typical for Streptococcus galloliticus.

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, unlike 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 group D streptococci.

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

The cultivation of a pure culture of the pathogen is carried out in meat and peptone broth. For infection, take 1-2 cm ³ culture of Streptococcus galloliticus and inoculate 80-100 ml of liquid nutrient medium and grow at 37 ° C for 12-14 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 .

To determine the morphology and tinctorial properties of the causative agent of pseudomonosis, fingerprints are prepared from the affected organs of the fallen animals, and they are stained using the Gram method. During microscopy of the preparations, under the immersion system of the microscope, straight or curved sticks 1.5–3.0 μm in size are visible, located singly, in pairs, in the form of chains, colored pink, characteristic of Ps. aeruginosa.

To determine the cultural properties of the causative agent of pseudomonosis, the selected culture is inoculated with meat - peptone broth, Petri dishes with meat - peptone agar (MPA), blood MPA and selective medium containing cetyltrimethyl ammonium bromide, on which other pseudomonads do not grow.

After 24 hours of incubation in a thermostat at a temperature of 37 ° C, a surface silver-white film in BCH is observed, a characteristic sign and turbidity of the medium. In Petri dishes with blood MPA, growth of grayish, translucent colonies surrounded by a greenish hemolysis zone is observed, which is typical for Pseudomonas aeruginosa.

Pure cultures of E. coli colibacteriosis, streptococcosis Streptococcus galloliticus and pseudomonosis Ps. aeruginosa is used to produce the vaccine.

The cultivation of pure cultures of pathogens is carried out separately in meat - peptone broth. For infection, take 5 cm ³ fresh clean culture of the causative agents of colibacteriosis E. coli, streptococcosis Streptococcus galloliticus and pseudomonosis Ps. aeruginosa per 1000 cm ³ meat - peptone culture medium with the addition of glucose to 0.2% concentration and grown at 37 ° C for 12-14 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.

Thus, the use for the manufacture of a vaccine associated against Escherichiosis, streptococcosis and pseudomonosis of pure cultures of pathogens E. coli, Str. galloliticus and Ps. aeruginosa, isolated from a local epizootic focus, provides a specific, highly immunogenic, associated vaccine to protect against these three dangerous infectious diseases of Escherichiosis, streptococcosis and cattle pseudomonosis. Separate growth of pathogens allows to obtain a concentration of E. coli, Str. galloliticus and Ps. aeruginosa at least 4 billion in 1 cm ³ for 12-14 hours of incubation. The use of formalin for inactivation in 0.1–0.4% final concentration makes it possible to suppress pathogenicity for 48–96 hours at a temperature of 37 ° С and maintain high immunogenicity during 12 months of storage. The vaccine obtained by the claimed method, has been tested. The rabbits were injected once with the received associated vaccine at a dose of 1 cm ³ , and after 12-15 days in vaccinated animals the formation of intense immunity against Escherichiosis, streptococcosis and pseudomonosis of cattle, lasting at least 9 months (observation period) is provided. After a single injection, the animals do not intramuscularly cause post-vaccination complications.

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

Claims (1)

A method of manufacturing a vaccine associated against Escherichiosis, streptococcosis and pseudomonosis in cattle, comprising obtaining antigens in the form of suspensions prepared from samples of pathological material taken from a patient and fallen cattle from a local epizootic focus, plating on differential diagnostic media, isolation of cells pure cultures of Escherichia coli Escherichiosis causative agents, Streptococcus streptococcosis, with their separate cultivation in meat and peptone broth with glucose added to 0.2% radios with a titer of 4-5 billion microbial cells in 1 cm ³ , separate inactivation of cells of pure cultures of Escherichia coli causative agents of Escherichiosis, streptococcosis Streptococcus formalin to a 0.4-0.5% final concentration at a temperature of 37 ° C for 72-96 hours with periodic stirring, mixing them in equal proportions, introducing into the mixture of inactivated cells pure cultures of adjuvant, which is used aluminum hydroxide in an amount of 20% by volume, thorough mixing and repeated thorough mixing before packing the final product, characterized in that cells of a pure culture of pathogens of pseudomonosis Pseudomonas aeruginosa and streptococcosis Streptococcus gallolyticus, which are separately grown in meat and peptone broth with the addition of glucose to 0.2% concentration with a titer of 4-5 billion microbial cells in 1 cm ³ , are additionally isolated separate formalin inactivation to 0.4-0.5% final concentration at 37 ° C for 72-96 hours with periodic stirring and cells of pure cultures of Escherichia coli, Escherichia coli pathogens, Pseudomonas aeruginosa pseudomonosis and streptococcus are mixed goat Streptococcus gallolyticus in equal proportions.