RU2404801C1 - Method for manufacturing of vaccine against colibacteriosis of rabbits - Google Patents

Abstract

FIELD: medicine, veterinary science.

SUBSTANCE: invention relates to veterinary science. Method consists in the fact that affected organs are picked from fallen rabbits from local epizootic focus, from which suspension is prepared. Inoculation is made onto differential-diagnostic media, pure culture Escherichia coli 035 is extracted, which is grown in meat-peptone broth. Concentration of microbial cells is increased up to titre of 5-6 billions in 1 cm³ and inactivated by introduction of formalin up to 0.4-0.5% final concentration. Maintained at the temperature of 37°C for 48-72 hours, then solution of aluminium hydroxide is added in amount of 20% to the volume, thoroughly mixed, packed and sealed.

EFFECT: method is simple in realisation, makes it possible to produce highly immunogenic inactivated vaccine.

1 tbl

Description

The invention relates to veterinary medicine, in particular to vaccines, and can be used in institutions involved in the design of biological products, as well as in enterprises of the biological industry.

A known method for producing a vaccine against colibacteriosis of piglets (RF patent for the invention No. 2022563, 05.26.88, MKI A61K 39/125, 39/135). This method involves the separate cultivation of strains of E. coli VGNKI No№305-37 / 10, 355-37 / 12, 357-37 / 12, 331-37 / 12, 359-37 / 12, 360-37 / 12, 299- 37/10, 330-37 / 11 in the Hottinger broth, mix the cultures in equal proportions, inactivate formalin and thiomersal, followed by adsorption on aluminum hydroxide. Used for the prevention of colibacillosis of piglets.

A known method of producing a vaccine for the prevention of streptococcosis nutria (RF patent for the invention No. 2099081 from 09/30/94, MKI A61K 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 remedy these shortcomings, in particular, to increase the efficiency of the method of manufacturing a vaccine against colibacteriosis of rabbits by introducing a new culture of the pathogen, new components and eliminating negative and side effects.

This object is achieved in that a method of manufacturing a vaccine against colibacteriosis of rabbits, including the production of antigen, inactivation, the introduction of an adjuvant, characterized in that the selection of the affected organs from dead rabbits from the local epizootic focus, from which the suspension is prepared, sowing on differential diagnostic environments is isolated pure culture of the pathogen grown culture of Escherichia coli O35 in meat-peptone broth with a titer of 5.6 billion microbial cells per 1 cm ^3, inactivated form by making ina strength to 0.4-0.5% final concentration, kept at a temperature of 37 ° C for 48-72 hours, then making the solution of aluminum hydroxide in an amount of 20% by volume, thoroughly mixed, packed and sealed.

The novelty of the proposed proposal is due to the fact that, in contrast to the known methods, organs from dead rabbits are taken during the period of illness and animal death from colibacteriosis, a suspension is prepared from pathological material, a pure culture is isolated by culture on differential diagnostic media, this allows you to select a pure culture of the pathogen. The use of meat-peptone culture medium when growing a pure culture of Escherichia coli O35 allows to obtain a concentration of microbial cells of at least 5-6 billion in 1 cm ³ . Inactivation of the culture by introducing formalin to 0.4-0.5% of the final concentration at a temperature of 37 ° C for 48-72 hours, reliably suppresses the pathogenicity of the pathogen, while maintaining immunogenicity. The method provides a harmless, highly immunogenic, specific vaccine for protecting rabbits against colibacteriosis.

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, concentrated polyvalent aluminum hydroxide approved by the General Directorate of Veterinary Medicine with the State Veterinary Inspection on 10.10.1991.

The proposed vaccine against colibacteriosis of rabbits is simple to implement, affordable and is industrially applicable.

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 guide zoopathogenic microorganisms "under the editorship of prof. Sidorova M.A. - Moscow: Kolos, 1995, pp. 196-201, as well as in the "Workshop on Veterinary Microbiology and Immunology" authors: Kostenko TS, Rodionova VB, Skorodumov DI - M .: Kolos, 2001, p. 219-222.

The essence of the proposed method consists in the selection of affected organs from dead rabbits from the local epizootic focus, from which the suspension is prepared, inoculation on differential diagnostic media, a pure pathogen culture is isolated, the culture of Escherichia coli O35 is grown in meat-peptone broth to concentration with a titer of 5-6 billion microbial cells in 1 cm ³ , inactivate by adding formalin to 0.4-0.5% final concentration, incubate at a temperature of 37 ° C for 48-72 hours, add a solution of aluminum hydroxide niya in an amount of 20% by volume, mix thoroughly, pack and seal.

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

To determine the morphology and tinctorial properties of the causative agent of colibacteriosis, smears are prepared - prints from the affected organs of the fallen nutria - by staining with a fat-free glass to a fresh section of the affected tissue and subsequent fixation over the flame of an alcohol lamp, they are stained using the Gram method. When microscopying the preparations under the immersion system of the microscope, thick sticks with rounded ends of red color, located singly, are observed, which is typical for Escherichia coli.

To determine the cultural properties of the causative agent of colibacteriosis, Nutria Escherichia coli make inoculated cultures from pathological material into meat-peptone broth (MPB), on Endo medium, meat-peptone agar (MPA) and incubate in an incubator at 37 ° C for 16-24 h. 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 slight precipitate breaking upon shaking. In Petri dishes, on Endo medium, raspberry-red colonies are observed to grow with a pink rim of 2-3 mm in diameter, characteristic of Escherichia coli.

When determining the biochemical activity of pure bacultures, crops are sown on differential diagnostic media with a different set of components. Escherichia 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 with 100% safety in the control. To confirm the specificity of the death of mice from the parenchymal organs of fallen mice, smears — fingerprints — are prepared and stained using the Gram method. When microscoping under the immersion system of the microscope, thick rods with rounded ends of red color, located singly, characteristic of Escherichia 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 Escherichia coli culture reacts with O35 coliserum and a flocculent precipitate (positive result) is seen with a negative control.

The cultivation of a pure culture of the pathogen Escherichia coli 035 is carried out in a meat-peptone culture medium. For infection, take 1-2 cm ^{3 of} fresh culture and sow 80-100 ml of liquid nutrient medium 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 the obtained raw materials is carried out by adding formalin of 0.4-0.5% final concentration, kept at a temperature of 37 ° C for 48-72 hours with periodic stirring, then a solution of aluminum hydroxide in an amount of 20% by volume is added and thoroughly mixed, Packed and corked.

Thus, the use of a pure culture of Escherichia coli O35 isolated from the affected organs of rabbits from the local epizootic foci for the preparation of a vaccine against colibacteriosis in rabbits allows one to obtain a specific, highly immunogenic vaccine to protect rabbits from colibacteriosis. Growing a pure culture of the pathogen in a meat-peptone nutrient medium allows to obtain a concentration of bacterial cells of at least 5-6 billion in 1 cm ³ for 12-14 hours of cultivation. The use of formalin for inactivation in a 0.4-0.5% final concentration allows suppressing pathogenicity for 48-72 hours at a temperature of 37 ° C and maintaining high immunogenicity for 12 months of storage. Adsorption of antigen on aluminum hydroxide allows 12-15 days after administration to rabbits to provide vaccines with the formation of intense immunity, lasting at least 9 months (observation period). After a single administration to nutria, intramuscularly does not cause post-vaccination complications. The results of the comparative characteristics of the vaccines are presented in 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. Pathogens strain Streptococcus zooepidemicus VGNKI No. K-DEPT the culture of the pathogen E.coli O35 isolated from the affected organs from dead rabbits from the local epizootic focus in colibacteriosis 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 48-72 hours at 37 ° C four. Adjuvant 3% solution of aluminum hydroxide in an amount of 10% by volume aluminum hydroxide solution 20% by volume 5. Post-vaccination complications in nutria after the introduction of the chromate vaccine, inflammatory processes no 6. Immunity duration 6 months 9 months

Thus, the proposed method of manufacturing a vaccine against colibacteriosis of rabbits has several advantages compared with the prototype.

Claims (1)

A method of manufacturing a vaccine against colibacteriosis of rabbits, including the production of antigen, inactivation, the introduction of an adjuvant, characterized in that the selection of the affected organs from the dead rabbits from the local epizootic focus, from which the suspension is prepared, inoculation on differential diagnostic media, a pure culture of Escherichia coli is isolated O35, which are grown in a meat-peptone broth, microbial cell concentration is adjusted to a titer of 6.5 billion. in 1 cm ^3, inactivated by formalin making up 0.4-0.5% ultimate strength concentra ation, maintained at a temperature of 37 ° C for 48-72 hours, then making the solution of aluminum hydroxide in an amount of 20% by volume, thoroughly mixed, packed and sealed.