RU2301076C1 - Method for preparing mixed vaccine against streptococcosis and enterococcus infection in nutria - Google Patents

Abstract

FIELD: biotechnology, microbiology.

SUBSTANCE: method involves selection of damaged organs from dead nutrias from local epizootic focus and preparing suspension from these organs. Then method involves inoculation in differential diagnostic media and isolation of pure cultures of pathogens. Cultures Streptococcus pneumoniae and Streptococcus faecalis are grown in beef-extract broth with addition glucose to final 0.2% concentration and with titer value 4-5 billions microbial cells/1 cm³ and inactivated by addition of formalin to final concentration 0.4-0.6% and kept at temperature 37°C for 72-96 h. Cultures are mixed in equal ratios, aluminum hydroxide solution is added in the amount 15% to volume, stirred thoroughly and sealed. Method provides preparing harmless, highly immune 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 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, Pseudomonas aeruginosa receive toxoid, 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 staphi okokkovy 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; nutria is not used to prevent infectious diseases.

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 glucose strains of Streptococcus zooepidemicus VGNKI No. K-DEPH, 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 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 technology for producing this vaccine is complex. Such a vaccine after administration to nutria causes post-vaccination complications (inflammation, abscesses, chromate).

The technical solution to 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 enterococcal nutria infection by introducing new pure cultures of pathogens, components, eliminating negative and side effects.

The solution to the problem is achieved by the fact that a method of manufacturing a vaccine associated against streptococcosis 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 on differential diagnostic media , pure cultures of pathogens are isolated, Streptococcus pneumoniae and Streptococcus fecalis cultures are separately grown in meat-peptone broth with glucose added up to 0.2% conc. Centerings 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, incubate 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 15% 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 affected organs from fallen nutria during the disease and the death of nutria from streptococcosis and enterococcal infection, prepare a suspension of pathological material, and isolate pure cultures of pathogens by sowing on differential diagnostic media. When separately grown, pure cultures of the pathogens Streptococcus pneumoniae and Streptococcus fecalis are used, meat-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. 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 enterococcal infection. The addition of aluminum hydroxide in an amount of 15% 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 streptococcosis 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 ., 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, however, these vaccines are not used for nutria.

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

For the manufacture of a vaccine associated against streptococcosis and enterococcal infection, nutria carry out the selection of affected organs from fallen nutria during the period of their illness with streptococcosis 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 causative agents of streptococcosis and 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 of the colony surrounded by a greenish hemolysis zone is observed, which is typical for the genus Streptococcus.

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 a culture of streptococci of serological group D, it is necessary to determine the variety of enterococci, since in addition to pathogenic Str. fecalis also includes Str. 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 Str. 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 the causative agents of Streptococcus Streptococcus pneumoniae and Enterococcal infection Str. fecalis is used to produce bacterial raw materials.

Pure cultures are grown separately in meat-peptone broth. For infection, take 5 cm ^{3 of} fresh pure cultures of causative agents of streptococcosis Streptococcus pneumoniae and enterococcal infection Str. fecalis 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 . Inactivation of the obtained raw materials is carried out by adding formalin of 0.4-0.6% of the final concentration, maintained 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 15% by volume is added to the baculture mixture and thoroughly mixed, packaged and sealed.

Thus, the use of pure cultures of Streptococcus pneumoniae and Streptococcus fecalis pathogens isolated from the local epizootic foci for the preparation of a vaccine associated with streptococcosis and enterococcal nutria infection allows the preparation of a more specific, highly immunogenic associated vaccine to protect against these two dangerous infectious diseases: streptococcosis and enter 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 within 12-14 hours of incubation. The use of formalin for inactivation in a 0.4–0.6% final concentration makes it possible to suppress pathogenicity for 72–96 hours at a temperature of 37 ° С and maintain high immunogenicity during 12 months of storage. 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 streptococcosis 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 (see 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 the pathogens Streptococcus pneumoniae and Streptococcus fecalis 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 15% 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 streptococcosis and enterococcal nutria infection in comparison with the existing prototype.

Claims (1)

A method of manufacturing a vaccine associated with streptococcosis and enterococcal nutria infection, 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 is carried out, pure cultures of pathogens are isolated, cultures are separately grown. Streptococcus pneumoniae and Streptococcus fecalis in meat-peptone broth supplemented with glucose to 0.2% concentration with a titer of 5.4 billion microbial cells per 1 cm ^3, made by inactivating I formalin to 0.4-0.6% of the 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 15% by volume, mix thoroughly, Packed and corked.