RU2292914C1 - Method for preparing associated vaccine aginst salmonellosis and enterococcal infection in nutrias - Google Patents

Abstract

FIELD: veterinary medicine.

SUBSTANCE: for manufacturing an associated vaccine it is necessary to select affected organs in dead nutrias from local epizootic focus for the purpose to prepare the suspension and fulfill inoculation onto differentially-diagnostic media. One should isolate pure cultures and separately grow the cultures of salmonellosis agents Salmonella typhimurium and enterococcal infection Streptococcus fecalis in beef-extract broth at addition of 0.2%-glucose at the titer being about 4-5 bln. microbial cells/cu. cm followed by inactivation due to applying formalin up to 0.4-0.6%-final concentration to be kept at 37°C for about 72-96 h. Cultures should be mixed at 1:2 ratio, then aluminum hydroxide solution should be applied at the quantity of 20% against the culture volume to be then thoroughly mixed, packed and sealed. The innovation enables to obtain safe, specific and high-immunogenic vaccine.

EFFECT: higher efficiency.

1 ex, 1 tbl

Description

The invention relates to veterinary medicine, in particular to a method for the manufacture of 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 A 61 K 39/125, 39/135). This method involves the separate isolation of protective antigens, from the Staphylococcus aureus strain No. B-243, the cytoplasmic antigen is isolated, from the toxin-producing strain of Pseudomonas aeruginosa NIIEM No. PA-7, Pseudomonas aeruginosa is received, from clinical strains of Proteus mirabilis No. 6, 8, 15, 50, 50 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 stafilo okkovy toxoid and aluminum hydroxide in an amount of 5-10 EU and 1-2 mg, respectively. The technology for producing the vaccine is very complicated; it is not used for the prevention of nutria.

There is a method of obtaining a vaccine against streptococcosis and pasteurellosis nutria (RF patent for the invention No. 2099083 from 09/30/94, MKI A 61 K 39/125, 39/135). This method involves 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, and the liquid is separated , inactivate with 0.3-0.4% formalin solution, combine them in equal proportions by volume and sequentially introduce aluminum hydroxide. This method allows to obtain a vaccine against streptococcosis and pasteurellosis nutria.

The technical solution to the problem of the invention is to remedy these shortcomings, in particular, to increase the specificity, efficiency of the method for manufacturing the associated vaccine against salmonellosis and enterococcal nutria infection by introducing new pure cultures of pathogens and 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 salmonellosis and enterococcal nutria infection, including obtaining antigens, inactivation, and adjuvant administration, selects affected organs from fallen nutria from the local epizootic focus, from which the suspension is prepared, inoculate onto differential diagnostic media , pure cultures are isolated, cultures of Salmonella typhimurium salmonellosis pathogens and Streptococcus fecalis enterococcal infections are separately grown in meat-peptone broth e 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 0.4-0.6% final concentration, incubated at a temperature of 37 ° C within 72-96 hours, mix the cultures in a ratio of 1: 2, then add a solution of aluminum hydroxide in an amount of 20% by volume of the culture, mix thoroughly, pack and seal.

The novelty of the proposed proposal is due to the fact that, in contrast to known methods, the selection of affected organs from fallen nutria during the disease and the death of nutria from salmonellosis and enterococcal infection is carried out, a suspension is prepared from pathological material, a pure culture is isolated by inoculation on differential diagnostic media. When separately grown, pure cultures of Salmonella typhimurium salmonellosis pathogens and Streptococcus fecalis enterococcal infection meat-peptone broth with glucose up to 0.2% are used and a microbial cell concentration of at least 4-5 billion in 1 cm ^{3 is} obtained. Formalin is inactivated to a 0.4-0.6% final concentration at 37 ° C for 72-96 hours and pure cultures are mixed in a ratio of 1: 2, which allows to completely suppress pathogenicity and maintain high immunogenicity against salmonellosis and enterococcal infection . 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 to protect nutria from salmonellosis and enterococcal infection.

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 The instructions for the manufacture and control of this vaccine were approved by the General Veterinary Administration of the Ministry of Agriculture of the USSR on March 28, 1980; 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, provides all the necessary information about the microorganisms of the genus Salmonella and the genus Streptococcus. However, for vaccines, 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." - M .: VO "Agropromizdat", 1990, as well as in the directory "Key to Zoopathogenic Microorganisms" edited by Professor Sidorov M.A. - M .: Kolos, 1995, pp. 201-204. In "Workshop on Veterinary Microbiology and Immunology" authors: Kostenko TS, Rodionova VB, Skorodumov DI - M .: “Kolos”, 2001, on pages 222-231.

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. - M .: Kolos, 1995, pp. 90-95.

The essence of the proposed method lies in the fact that they carry out the selection of affected organs from fallen nutria from the local epizootic focus, prepare a suspension of pathological material, sow on differential diagnostic media, isolate pure cultures of pathogens of infectious diseases of salmonellosis and enterococcal infection, separate pure cultures of pathogens Salmonella typhimurium and Streptococcus fecalis in meat-peptone broth with glucose added to 0.2% concentration with a titer of 4-5 billion microbial cells in 1 cm ³ , inactivate by introducing formalin to 0.4-0.6% of the final concentration, incubate at a temperature of 37 ° C for 72-96 hours, mix the cultures in a ratio of 1: 2, then add a solution of aluminum hydroxide in an amount of 20% to the volume of culture, thoroughly mix, pack and seal.

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

For the manufacture of a vaccine associated with salmonellosis and enterococcal infection, nutria carry out the selection of affected organs from fallen nutria during the period of illness with their salmonellosis and enterococcal infection, 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 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 thermostat at 37 ° C for 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 characteristic metallic luster on bismuth-sulfite 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 causative agent of salmonellosis is characteristic of representatives of the genus Salmonella (ferments glucose, beckons, 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- and H-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 enterococcal infection, 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 and arranged in pairs, colored in purple, characteristic of the genus Streptococcus. To determine the cultural properties of the causative agent of enterococcal infection, 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 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 colonies surrounded by a greenish hemolysis zone is observed, which is typical for Streptococcus fecalis.

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.

When isolating the culture of streptococci of serological group D, it is necessary to determine the variety of enterococci, since in addition to pathogenic Streptococcus fecalis, this group also includes Streptococcus faecium, which is an obligate inhabitant of the intestines of animals and humans. To do this, use a differential diagnostic environment. On enterococcal differential diagnostic medium after 14-18 hours of incubation, the growth of cherry red colonies is observed, which is typical for the causative agent of enterococcal infection Streptococcus fecalis. 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 Salmonella typhimurium salmonellosis pathogens and Streptococcus fecalis enterococcal infection isolated in this way are used to produce bakery.

The cultivation of pure cultures of pathogens is carried out separately in meat-peptone broth. For infection, take 5 cm ³ fresh pure culture of the pathogen Salmonella Salmonella typhimurium 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 0.4-0.6% final concentration, kept at a temperature of 37 ° C for 72-96 hours with periodic stirring.

For infection, take 5 cm ^{3 of a} pure culture of the causative agent of enterococcal infection Streptococcus fecalis 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 a ratio of 1: 2. 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 Salmonella typhimurium and Streptococcus fecalis pathogens isolated from the local epizootic focus for the manufacture of a vaccine associated with salmonellosis and enterococcal nutria infection allows the preparation of a more specific, highly immunogenic associated vaccine to protect against these two dangerous infectious diseases: salmonellosis and enterococcus 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 two 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 salmonellosis and enterococcal infection 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 baculture of Salmonella typhimurium salmonellosis causative agents and Streptococcus fecalis enterococcal infections of the nutria isolated from the local epizootic focus 2. Freeze-thaw, separate the liquid fraction Necessarily carried out Not carried out 3. Inactivation of the bacterial mass 0.3-0.4% formalin solution, 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 salmonellosis and enterococcal nutria infection in comparison with the existing prototype.

Claims (1)

A method of manufacturing a vaccine associated against salmonellosis and enterococcal nutria infection, including 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 are isolated, and cultures of Salmonella typhimurium and enterococcal infections are separately grown. Streptococcus fecalis in meat-peptone broth supplemented with glucose to 0.2% concentration with a titer of 4.5 billion. microbial cells per 1 cm ^3, inactivates they are removed by adding formalin to a 0.4-0.6% final concentration, kept at a temperature of 37 ° C for 72-96 hours, the cultures are mixed in a ratio of 1: 2, then a solution of aluminum hydroxide is added in an amount of 20% by volume cultures, mix thoroughly, pack and seal.