RU2388489C1 - Method for preparing vaccine associated against pseudomonosis and enterococcus infection of nutrias - Google Patents

Abstract

FIELD: medicine, veterinary science.

SUBSTANCE: invention concerns veterinary medicine. The method involves production of the antigen, inactivation and addition of adjuvant. Target affected organs of fallen nutrias are taken from a local epizootic nidus, and suspension is prepared from them. Inoculation in differentially diagnostic media is carried out subsequently, pure cultures of the germs are evolved, cultures Pseudomonas aeruginosa and Enterococcus faecalis are separately grown in meat infusion broth with glucose added up to 0.2% concentration with titre of 4-5 milliard microbe cells per 1 cm³. The material is inactivated by adding formalin up to 0.4-0.5% final concentration and then exposed to 37°C for 72-96 hours. The cultures are mixed in equal proportion. Aluminium hydroxide (20% by volume) is added to the culture. The components are thoroughly mixed, packed and sealed.

EFFECT: method is simple and allows of obtaining highly immunogenic specific 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, 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 receive Pseudomonas aeruginosa, from clinical strains of 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 stafilok CCSQ 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.

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 involves separate cultivation in a liquid nutrient medium with the addition of glucose of the strains of Streptococcus zooepidemicus VGNKI No. K-DEPT, Pasteurella multosida VGNKI No. 2394 and Pasteurella multocida VGNKI No. 1015 for 18-24 hours, the resulting culture medium is 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 technical solution to the problem of the invention is to eliminate the above disadvantages, in particular, increasing the specificity, efficiency of the method of manufacturing the associated vaccine against pseudomoniasis and enterococcal nutria infection by introducing new pure cultures of pathogens, components, excluding negative and side effects.

The solution to the problem is achieved in that a method of manufacturing a vaccine associated against pseudomoniasis and enterococcal nutria infection, including obtaining antigen, inactivation, and adjuvant administration, is characterized in that the affected organs are selected from fallen nutria from the local epizootic focus, from which the suspension is prepared, inoculation is made on differential diagnostic environments, pure cultures are isolated, cultures of Pseudomonas aeruginosa and Enterococcus faecalis are separately grown in meat-peptone broth with the addition of glucose up to 0.2% centration with a titer of 4-5 billion. microbial cells per 1 cm ^3, inactivated by formalin making up 0.4-0.5% strength final concentration, kept at a temperature of 37 ° C for 72-96 hours, cultures were mixed in equal ratios, 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 known methods, the selection of affected organs from fallen nutria during the disease and the death of nutria from pseudomonosis 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 the pathogens Pseudomonas aeruginosa and Enterococcus Enterococcus faecalis infection and 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 0.4-0.5% 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 pseudomonosis 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 dose of 1 cm ³ nutrias 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 pseudomonosis 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.

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 of the genus Streptococcus. However, these vaccines are not used for nutria.

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, p. 522- 525.

Methods of isolation and characterization of streptococci are presented in the Methodological Instructions for Laboratory Diagnosis of Animal Streptococcosis, approved on September 25, 1990, by the Main Veterinary Administration with the State Inspectorate under the State Commission of the USSR Council of Ministers for Food and Procurement, and also in the reference book Zoopathogenic Microorganism Guidebook under Edited by 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, from which the suspension is prepared, inoculum is made on differential diagnostic media, pure cultures of pathogens are isolated, cultures of Pseudomonas aeruginosa and Enterococcus faecalis are separately grown in peptone meat broth supplemented with glucose to 0.2% concentration with a titer of 4.5 billion. microbial cells per 1 cm ^3, inactivated by formalin making up 0.4-0.5% strength final concentration, kept at evap e 37 ° C for 72-96 hours, cultures were mixed in equal proportions, then making the solution of aluminum hydroxide in an amount of 20% by volume, thoroughly mixed, packed and sealed.

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

For the manufacture of the vaccine associated against pseudomoniasis and enterococcal infection, nutrias carry out the selection of the affected organs from fallen nutria during the period of their disease with pseudomoniasis 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 causative agent of pseudomonosis, fingerprint smears are prepared from the affected organs of the fallen nutria, 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 seen, arranged singly, in pairs, in the form of chains, colored in pink, characteristic of Pseudomonas aeruginosa.

To determine the cultural properties of the causative agent of pseudomonosis, crops of the isolated culture are made from the material in the meat-peptone broth, in 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.

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. During microscopy of preparations, under the immersion system of the microscope, small cocci are observed in the form of chains and arranged in pairs, colored in purple, characteristic of the genus Enterococcus. To determine the cultural properties of the causative agent of enterococcal infection, the selected culture is inoculated from the material into meat-peptone broth with glucose added 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 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 Enterococcus faecalis.

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 streptococci.

When isolating a culture of streptococci, it is necessary to determine the variety of enterococci, since in addition to pathogenic Enterococcus faecalis, 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 Enterococcus faecalis, the pathogen of enterococcal infection. 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 pseudomonosis Pseudomonas aeruginosa and enterococcal Enterococcus faecalis pathogens 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 causative agent of the pseudomonosis Pseudomonas 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.

For infection, take 5 cm ^{3 of a} pure culture of the causative agent of the enterococcal infection Streptococcus faecalis 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 of pure cultures of the pathogens Pseudomonas aeruginosa and Enterococcus faecalis isolated from the local epizootic foci for the preparation of a vaccine associated with pseudomonosis and enterococcal infection of nutria makes it possible to obtain a more specific, highly immunogenic associated vaccine to protect against these two dangerous infectious diseases: pseudomonosis and enterococcal 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 0.4-0.5% final concentration allows to suppress pathogenicity and maintain high immunogenicity for 12 months of storage for 72-96 hours at a temperature of 37 ° C. 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 pseudomonosis and enterococcal infection, lasting at least 9 months (observation period). After a single injection, nutria intramuscularly does not cause post-vaccination complications in them (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 zooepidemicus VGNKI No. K-DEPT, Pasteurella multosida VGNKI No. 2394 and Pasteurella multocida VGNKI No. 1015 Pure baculture of pathogens of pseudomonosis Pseudomonas aeruginosa and enterococcal nutria infection Enterococcus faecalis isolated from the local epizootic focus 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, incubated for 44-50 hours 0.4-0.5% 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 vaccine, lameness, 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 pseudomoniasis and enterococcal nutria infection in comparison with the existing prototype.

Claims (1)

A method of manufacturing a vaccine associated against pseudomoniasis and enterococcal infection of nutria, including obtaining antigen, inactivation, the introduction of an adjuvant, characterized in that the selection of the affected organs from the fallen nutria from the local epizootic focus, from which the suspension is prepared, inoculation on the differential diagnostic environment is isolated pure pathogen cultures; separately cultivate Pseudomonas aeruginosa and Enterococcus faecalis cultures 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 adding formalin to 0.4-0.5% final concentration, incubated at 37 ° C for 72-96 hours, mix the cultures in equal proportions, then add the solution aluminum hydroxide in an amount of 20% by volume, thoroughly mixed, packaged and sealed.