RU2288002C1 - Method for preparing vaccine against enterococcus infection in nutrias - Google Patents

Abstract

FIELD: veterinary medicine.

SUBSTANCE: the suggested vaccine as an antigen contains the suspension of cells of pure culture of Streptococcus fecalis agent obtained due to selecting affected organs in dead nutrias from local epizootic focus to prepare the suspension followed by inoculation onto differentially-diagnostic media; then one should isolate pure culture of Streptococcus fecalis agent to be grown in beef-infusion-peptone broth at addition of 0.2%-glucose to achieve the concentration of about 5-6 bln. microbial cells/cu. cm followed by inactivation due to introducing formalin up to 0.4-0.6%-final concentration to be kept at 37 C for about 72-96 h and aluminum hydroxide at the quantity of 20% against the volume of the culture to be thoroughly mixed, packed and sealed. The vaccine is safe and highly efficient.

EFFECT: higher efficiency.

1 ex, 1 tbl

Description

The invention relates to veterinary medicine, in particular to a method for manufacturing a vaccine, 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.

There is a method of obtaining a vaccine against calves Escherichiosis (RF patent for the invention No. 9302024613, MKI A 61 K 39/125, 39/135, 04/20/93). This method involves the cultivation of toxigenic E. Coli strains on Hottinger medium for 5-7 days, the microbial mass is separated from the toxin on filters with sterilizing plates, then the toxin is inactivated in the filtrate with formalin in a 0.3-0.4% concentration at 37 ° C for 10-12 days, then the resulting toxoid is precipitated with a 10% solution of alum-potassium alum at a rate of 1% for the entire volume, after thawing for 2-3 days, the supernatant is removed, the resulting complex toxoid is mixed in equal proportions with the antigen, received th from strains after growth of Escherichia allowance broth cultures at 37 ° C for two days, and is performed flush microbial mass saline containing 0.3-0.4% with the addition of formalin to a concentration of 50-60 billion microbial bodies in 1 cm ³ by adding to flush hydrogen peroxide to its content up to 0.15-0.25%, moreover, the mixture of toxoid and antigen is diluted with sterile distilled water to a concentration of 5 billion microbial bodies. This method allows to obtain a vaccine for the prevention of calves escherichiosis.

A known method of obtaining a vaccine for the prevention of streptococcosis of nutria (RF patent for the invention No. 2099081 from 09/30/94, MKI A 61 K 39/125, 39/135). This method involves the cultivation of a strain of Streptococcus zooepidemicus VGNKI No. K-DEPT in a liquid nutrient medium with glucose added to achieve a concentration of 5-6 billion microbial cells in 1 cm ³ , then subjected to freezing and thawing, the liquid fraction is separated, 0.3- A 0.4% formalin solution is kept for 44-50 hours and a 3% solution of aluminum hydroxide is added in an amount of 10% by volume of the culture. The technology for producing the vaccine is complex, it is used to prevent streptococcosis of 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 a vaccine against enterococcal nutria infection by introducing a new culture of the pathogen, components, excluding negative and side effects.

The solution to the problem is achieved in that a method of manufacturing a vaccine against enterococcal nutria infection, including receiving 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, and differential diagnostic tests are inoculated environment, isolated pure culture of the pathogen Streptococcus fecalis and grown in a meat-peptone broth supplemented with 0.2% glucose to achieve a concentration of 5-6 billion microbial cells per 1 cm ^3, inactivated comfort by making up 0.4-0.6% formalin cent final concentration, kept at a temperature of 37 ° C for 72-96 hours, then aluminum hydroxide is introduced in an amount of 20% of the culture volume, thoroughly mixed, packed and sealed.

The novelty of the proposed proposal is due to the fact that, in contrast to the known methods, the selection of affected organs from fallen nutria during the disease and the death of nutria from enterococcal infection, from which the suspension is prepared, isolates a pure culture by sowing on differential diagnostic media. When growing a pure culture of Streptococcus fecalis in meat-peptone broth with the addition of 0.2% glucose, it is possible to obtain a concentration of microbial cells of at least 5-6 billion in 1 cm ³ . The method provides a harmless, specific, highly immunogenic vaccine to protect nutria from 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: 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 USSR 04/26/1984; instructions for the manufacture and control of a vaccine against bradzot, infectious enterotoxemia, malignant edema of sheep and lambs dysentery, multivalent concentrated aluminum hydroxide, approved by the General Directorate of Veterinary Medicine with the State Veterinary Inspection on 10.10.1991.

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 at 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 during the epizootic period of enterococcal infection, prepare a suspension of pathological material, make crops on differential diagnostic media, incubate at 37 ° C for 14-18 hours and isolate a pure culture causative agent of enterococcal infection. The cultivation of a pure culture of Streptococcus fecalis is carried out in a meat-peptone nutrient medium with the addition of 0.2% glucose to achieve a concentration of microbial cells of 5-6 billion in 1 cm ³ , inactivate formalin to 0.4-0.6% of the final concentration, withstand at a temperature of 37 ° C for 72-96 hours, aluminum hydroxide is added in an amount of 20% by volume of the culture, thoroughly mixed, packaged and sealed.

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

In the manufacture of a vaccine against enterococcal infection, nutria carry out the selection of affected organs from fallen nutria during the disease and death from enterococcal infection, prepare a suspension from pathological material and conduct bacteriological studies. To determine the morphology and tinctorial properties of the pathogen, 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, the selected culture is inoculated from the material in the meat-peptone broth with the addition of 0.2% glucose and 10% inactivated horse serum (MPB), in Petri dishes with meat-peptone agar (MPA) with the addition of 0, 2% glucose 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 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 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 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.

The cultivation of a pure culture of Streptococcus fecalis is carried out in meat-peptone broth pH 7.6-7.8. For infection, take 5 ml of fresh fecal streptococcus baculture per 1000 ml of liquid nutrient medium with the addition of 0.2% glucose 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 5-6 billion in 1 cm ³ . Inactivation of bakery is carried out by adding formalin of 0.4-0.6% final concentration, kept at a temperature of 37 ° C for 72-96 hours with periodic stirring, then mix and add a 3% solution of aluminum hydroxide in an amount of 20% to volume and mix thoroughly, packaged and corked.

Thus, the use of pure Streptococcus fecalis culture isolated from the local epizootic foci for the preparation of a vaccine against enterococcal infection of nutria allows one to obtain a more specific, highly immunogenic vaccine to protect against enterococcal infection. The growth of the pathogen with a content of 0.2% glucose allows to obtain a concentration of bacterial cells of at least 5-6 billion in 1 cm ³ during 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 for 12 months of storage. Adsorption of antigen on aluminum hydroxide allows 12-15 days after a single injection of 1 cm ³ nutria to provide vaccines with the formation of intense immunity against 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 1).

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

Table 1.
Comparative characteristics of the manufacture of the vaccine according to the proposed method and prototype No. p / p Features Prototype The proposed method one. The causative agent of enterococcal infection Strain Streptococcus zooepidemicus VGNKI No. K-DEP Culture of Streptococcus fecalis isolated from affected organs from fallen nutrias from a local epizootic focus in case of enterococcal infection 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 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, the data in table 1 show the advantages of the proposed method for the manufacture of vaccines against enterococcal nutria infection in comparison with the existing prototype.

Claims (1)

A method of manufacturing a vaccine against enterococcal nutria infection, including antigen production, inactivation, adjuvant administration, characterized in that the selection of affected organs from fallen nutria from the local epizootic focus, from which the suspension is prepared, inoculation on differential diagnostic media, isolating a pure pathogen culture Streptococcus fecalis and grown in meat-peptone broth with the addition of 0.2% glucose to achieve a concentration of microbial cells of 5-6 billion in 1 cm ³ , inactivated by applying formalin to 0 , 4-0.6% final concentration, kept at a temperature of 37 ° C for 72-96 hours, then aluminum hydroxide is added in an amount of 20% by volume of the culture, mixed thoroughly, packed and sealed.