RU2301077C1 - Method for preparing mixed vaccine against coli-bacteriosis, salmonellosis and streptococcosis in nutria - Google Patents

Abstract

FIELD: biotechnology, microbiology.

SUBSTANCE: invention relates to a method for preparing harmless, specific, highly immune, inactivated, mixed vaccine against coli-bacteriosis, salmonellosis and streptococcosis in nutria wherein method involves using antigens, inactivation and addition of adjuvant. Method involves selection of damaged organs from dead nutrias in local epizootic focus and preparing suspension from these organs followed by inoculation in differential diagnostic media. Pure cultures of coli-bacteriosis, salmonellosis and streptococcosis pathogens are isolated. Cultures Escherichia coli, Salmonella typhimurium and Streptococcus pneumoniae are grown separately in beef-extract broth with addition of 0.2% of glucose and with titer value 4-5 billions microbial cells/1 cm³. Inactivation is carried out by addition of formalin in the final concentration 0.4-0.6%. Then cultures are kept at temperature 37°C for 72-96 h and mixed their in equal ratio. Then aluminum hydroxide solution is added in the amount 20% to volume, stirred thoroughly, packaged and sealed. Method provides preparing harmless, immunogenic and highly specific vaccine.

EFFECT: improved preparing method.

1 tbl, 1 ex

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 obtaining a multivalent vaccine against intestinal infections (RF patent for the invention No. 2080875 from 03/18/92, MKI AK61K 39/125, 39/135). This method of obtaining a multivalent vaccine involves the separate cultivation of cultures of Salmonella typhi, Salmonella paratyphi B, Shigella Flexneria, Shigella Sonne, the separation of biomass from the nutrient medium, the extraction of antigens is carried out with water-saline solution by shuttling in the presence of an antibiotic and finely divided glass for 1-1.5 h, the extracts obtained are centrifuged, supernatants are purified and after mixing the desired product is obtained in the form of a mixture of cell walls containing O-antigens of the cultures used, with flagella containing H-antigen nas, and soluble Salmonella Vi antigens. The technology for producing this vaccine is very complex and is not used to prevent infectious diseases of nutria.

A known method of obtaining a vaccine against streptococcosis and pasteurellosis nutria (RF patent for the invention No. 2099083 from 09/30/94, MKI AK61K 39/125, 39/135). This method provides for separate cultivation in a liquid nutrient medium with the addition of glucose of the strains of Streptococcus zooepidemicus VGNKI No. K-DEPH, Pasteurella multosida VGNKI No. 2394 and Pasteurella multocida VGNKI No. 1015 for 18-24 hours, the obtained culture mediums are subjected to freezing-thawing, the liquid is separated , inactivate 0.3-0.4% formalin solution, combine them in equal proportions by volume and sequentially make aluminum hydroxide. This method allows to obtain a vaccine against streptococcosis and pasteurellosis nutria. The technology for producing this vaccine is complex. Such a vaccine after use in animals causes post-vaccination complications, the duration of immunity is 6 months. This vaccine protects nutria only from streptococcosis and pasteurellosis.

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 colibacteriosis, slammonellosis and streptococcosis in nutrias by introducing new pure cultures of pathogens, components, excluding negative and side effects.

The solution to the problem is achieved by the fact that a method of manufacturing a vaccine associated against colibacteriosis, salmonellosis and streptococcosis of nutria, including the production of antigens, inactivation, the introduction of an adjuvant, consists in the selection of affected organs from fallen nutria from the local epizootic focus, from which a suspension is prepared, made sowing on differential diagnostic media, pure cultures of pathogens of colibacteriosis, salmonellosis and streptococcosis are isolated, cultures of Escherichia coli, Sal are separately grown monella typhimurium, Streptococcus pneumoniae in meat and peptone broth with glucose up to 0.2% concentration with a titer of 4-5 billion microbial cells in 1 cm ³ , inactivate by adding formalin to 0.4-0.6% final concentration , kept 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, in contrast to the known methods, they carry out the selection of the affected organs from fallen nutria during the disease and the death of the nutria from colibacteriosis, salmonellosis and streptococcosis, prepare a suspension of pathological material, isolate pure cultures of pathogens by sowing on differential diagnostic media. Used for separate cultivation of pure cultures of Escherichia coli, Salmonella typhimurium, Streptococcus pneumoniae, meat and peptone broth with glucose added up to 0.2% and a microbial cell concentration of at least 4-5 billion in 1 cm ^{3 is} obtained. The pathogen cultures are inactivated by formalin to a 0.4-0.6% final concentration at 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 colibacteriosis, salmonellosis and streptococcosis. The addition of aluminum hydroxide in an amount of 20% to the volume of the culture allows 12-15 days after a single injection at a dose of 1 cm ³ nutria to provide the vaccinated with the formation of intense immunity against two infections. The method provides a harmless, highly immunogenic, more specific vaccine for protecting nutria from colibacillosis of salmonellosis and streptococcosis.

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.

With regard to 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: formolvac vaccine against paratyphoid (salmonellosis) calves TU 46-21-166-75, approved 10.05.75 g ., instructions for the manufacture and control of this vaccine approved by the General Directorate of Veterinary of the Ministry of Agriculture of the USSR 03/28/80; vaccine against paratyphoid (salmonellosis) piglets TU 46-21-952-74, approved July 15, 74, instructions for the manufacture and control of this vaccine approved by the General Directorate of Veterinary of the Ministry of Agriculture of the USSR 10.23.96; associated vaccine against salmonellosis, pasteurellosis and enterococcal infection of piglets TU 10.09-62-90, approved on 08.18.90, the instruction for the manufacture and control of this vaccine was approved by the General Directorate of Veterinary Medicine on 16.07.90; vaccine against enterococcal infection of calves, lambs and piglets TU 10.09-91-90, approved 15.12.90; instructions for the manufacture and control of polyvalent aluminum hydroxide formol vaccine against colibacteriosis (Escherichiosis) of piglets, calves and lambs, approved by the GUV of the Ministry of Agriculture of the USSR on 04/26/1984, however, these vaccines are not used for nutria.

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 reference book "Determinant of zoopathogenic microorganisms" edited by Professor Sidorov MA, Moscow - Kolos, 1995, pp. 201-204, in the "Workshop on Veterinary Microbiology and Immunology" Authors: Kostenko TS, Rodionova VB, Skorodumov DI, M .: Kolos, 2001, pp. 222-231.

Methods for isolating a pure culture of the causative agent of colibacteriosis and 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 reference book “Determinant of 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, 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, as well as in the reference book “Identifier of zoopathogenic microorganisms” Professor Sidorov M.A., Moscow. - Kolos, 1995, pp. 90-95.

The essence of the proposed method lies in the fact that they carry out the selection of the affected organs from fallen nutria from the local epizootic focus, prepare a suspension of pathological material, make inoculation on differential diagnostic media, isolate pure cultures of pathogens of infectious diseases of colibacteriosis, salmonellosis and streptococcosis, clean cultures are separately grown. Escherichia coli, Salmonella typhimurium and Streptococcus pneumoniae in meat and peptone broth with glucose added to 0.2% concentration with a titer of 4-5 billion microbial cells in 1 cm ³ , inactivate by adding formalin to 0.4-0.6% of the final concentration, incubated 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 % to the volume of culture, mix thoroughly, pack and seal.

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

For the manufacture of a vaccine associated against colibacteriosis, salmonellosis and streptococcosis, nutria, the affected organs are selected from fallen nutria during the period of their disease with colibacteriosis, salmonellosis and streptococcosis, 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 colibacteriosis, fingerprint smears are prepared from the affected organs of the fallen nutrias by touching a fresh cut of the affected tissue with defatted glass and subsequent fixation over the flame of the spirit 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 colibacteriosis of E. coli nutria, the selected culture is inoculated from pathological material into meat-peptone broth (MPB), Endo medium, meat-peptone agar (MPA) and incubated in an incubator at 37 ° C for 16-24 hours. On Petri dishes with MPA, wet colonies grow with smooth edges and a smooth gray surface. 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, the growth of raspberry-red colonies with a pink rim of 2-3 mm in diameter, characteristic of E. coli, is observed.

When determining the biochemical activity of pure bacultures, crops are made 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 the fallen mice, 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 pathogen of salmonellosis, fingerprint smears are prepared from the affected organs of the fallen nutria, and they are stained using the Gram method. When microscopying the preparations under the immersion system of the microscope, thin red rods are observed, located singly, characteristic of the genus Salmonella.

To determine the cultural properties of the causative agent of salmonellosis nutria, the selected culture is inoculated from pathological material into meat-peptone broth (MPB), on differential diagnostic media (Endo, Levina, bismuth sulfite agar), on meat-peptone agar (MPA) and incubated in an incubator at 37 ° C in within 16-24 hours. Smooth, transparent, colorless colonies with even edges grow on Petri dishes with MPA. In test tubes with BCH, diffuse turbidity of the nutrient medium is observed. Transparent colorless colonies are observed in Petri dishes, on Endo medium, bluish on Levin medium, black colonies with a characteristic metallic luster on bismuth sulphite agar, which is typical for the genus Salmonella.

When determining the biochemical activity of pure cultures, crops are sown on differential diagnostic media with a different set of carbohydrates and components.

The isolated pure culture of the pathogen of salmonellosis, characteristic of representatives of the genus Salmonella, ferments glucose, mannitol, does not utilize lactose, sucrose, does not break down urea, does not form indole, and does not dilute gelatin.

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 the genus Salmonella.

To establish the serotypic affiliation of the isolated pure Salmonella culture, an agglutination reaction (RA) was placed on glass according to the generally accepted method. The isolated pure culture was tested with O-complex and monorecentric O- and H-agglutinating sera in the agglutination reaction. To do this, the selected pure culture is grown on a beveled MPA for 18-24 hours at a temperature of 37 ° C. RA on glass is placed with each of the O-complex sera in series until a positive result with two sera is obtained. A positive RA result is observed in the form of hard-breaking flakes with an isolated culture of Salmonella typhimurium.

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 and 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 glucose up 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 pneumoniae.

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 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 Escherichia coli colibacteriosis, Salmonella salmonella typhimurium salmonellosis and Streptococcus pneumoniae streptococcosis pathogens isolated in this way are used to produce bakery. The cultivation of pure cultures of pathogens of colibacteriosis, salmonellosis and streptococcosis is carried out separately in meat and peptone broth.

Upon receipt of a pure culture of the causative agent of colibacteriosis, 1-2 cm ^{3 of} fresh E. coli culture are taken for infection, 80-100 cm ^{3 of} liquid nutrient medium are inoculated 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 5-6 billion in 1 cm ³ . The inactivation of the obtained raw materials is carried out by introducing formalin of 0.5-0.6% final concentration, kept at a temperature of 37 ° C for 72-96 hours with periodic stirring.

Upon receipt of a pure culture of the pathogen of salmonellosis for infection, take 5 cm ³ fresh pure culture of the pathogen of salmonellosis Salmonella typhimurium per 1000 cm ³ meat-peptone nutrient 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 0.4-0.6% final concentration, kept at a temperature of 37 ° C for 72-96 hours with periodic stirring.

Upon receipt of a pure culture of the causative agent of streptococcosis for infection, take 5 cm ^{3 of a} pure culture of the causative agent of streptococcosis Streptococcus pneumoniae 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 0.4-0.6% 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 mixture of bacultures and thoroughly mixed, packaged and sealed.

Thus, the use of pure cultures of Escherichia coli, Salmonella typhimurium, and Streptococcus pneumoniae pathogens isolated from the local epizootic foci for the preparation of a vaccine associated with salmonellosis, colibacteriosis, and streptococcosis of nutria allows obtaining a more specific, highly immunogenic, associated vaccine to protect against these three dangerous : colibacteriosis, salmonellosis and streptococcosis nutria. Separate growth of pathogens with a content of 0.2% glucose 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 0.4-0.6% of the final concentration allows to suppress pathogenicity for 72-96 hours at a temperature of 37 ° C and maintain high immunogenicity for 12 months of storage. The adsorption of three antigens on aluminum hydroxide allows 12-15 days after a single injection of 1 cm ³ nutria to provide the vaccinated nutria with the formation of intense immunity against colibacteriosis, salmonellosis and streptococcosis lasting at least 9 months (observation period). After a single administration to nutria, intramuscularly does not cause post-vaccination complications in them (table).

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

Table
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 zooepidemicus VGNKI No. K-DEPT, Pasteurella multosida VGNKI No. 2394 and Pasteurella multocida VGNKI No. 1015 Pure bacterial cultures of Escherichia coli colibacteriosis, Salmonella typhimurium salmonellosis and Streptococcus pneumoniae streptococcosis isolated from local epizootic foci 2. subjected to freezing-thawing, the liquid fraction is separated Necessarily carried out Not carried out 3. Inactivation of the bacterial mass 0.3-0.4% formalin solution is incubated for 44-50 hours 0.4-0.6% formalin concentration for 72-96 hours at a temperature of 37 ° C four. Adjuvant 3% solution of aluminum hydroxide in an amount of 10% by volume aluminum hydroxide 20% by volume 5. Post-vaccination complications In nutria, after administration of the chromate vaccine, inflammatory processes No 6. Immunity duration 6 months 9 months

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

Claims (1)

A method of manufacturing a vaccine associated against colibacteriosis, salmonellosis and streptococcosis of nutria, which consists in the selection of affected organs from fallen nutria from the local epizootic focus, from which the suspension is prepared, inoculation on differential diagnostic media, pure cultures of causative agents of colibacteriosis, salmonellosis and streptococcosis, cultures of Escherichia coli, Salmonella typhimurium and Streptococcus pneumoniae are separately grown in meat-peptone broth with glucose added to 0.2% concentration with t Trom 4-5 billion microbial cells per 1 cm ^3, inactivated by formalin making up 0.4-0.6% strength final concentration, kept at a temperature of 37 ° C for 72-96 hours, cultures were mixed in equal proportions, then make a solution of aluminum hydroxide in an amount of 20% by volume, mix thoroughly, pack and seal.