RU2432174C1 - Method for producing colibacillosis anatoxin - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, and can be used for creation of vaccine preparations for colibacillosis in animals. A method for producing colibacillosis anatoxin provides sampling of epizootic Escherichia coli strains able to produce a thermolabile, thermostable and shiga like toxin, separate cultivation on a nutritious broth for 6-7 days, culture inactivation by adding formalin to the concentration 0.3-0.4 % at temperature 37°C for 14 days and mixing in equal volumes, and separation of a bacterial mass by means of sterilisation filtration.

EFFECT: invention provides more efficient prevention of colibacillosis in animals.

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Description

The invention relates to biotechnology and can be used to create vaccines against colibacillosis of animals.

An important link in the system of measures for the prevention of colibacteriosis, the causative agent of which is pathogenic Escherichia coli, is the immunization of animals with specific vaccines. It was found that the most effective vaccines are those that contain inactivated exotoxins of the pathogen (Biological preparations for the specific prophylaxis and treatment of animal escherichiosis / M.K. Pirozhkov // Diss. Doc. Vet. Sciences. - Moscow, 2002. - 298 p.). Currently, all known vaccines include only two types of toxins of pathogenic Escherichia: thermolabile (LT) and thermostable (ST), although we have found that often (in 21.5% of cases) in the development of the pathological process in young animals plays shigapodobny (STX) toxin (Terekhov V.I. et al. Antigenic composition and pathogenic properties of E. coli strains isolated from calves and piglets in the Krasnodar Territory // Russian Veterinary Journal. Farm Animals, 2008, No. 4. - P.6-8) . Therefore, the inclusion of a specific drug for the prevention of colibacillosis of this type of toxin is also necessary.

Known vaccine Koli-Vak against escherichiosis of animals (RF patent for the invention No. 2043771, 1995). The vaccine includes various Escherichia antigens: somatic - 078, 0141; adhesive proteins - K88, K99, 987P, F41; capsular polysaccharide - K80, K87, TL, TS-toxoids. The authors believe that the components of the vaccine provide the synthesis of specific antibodies in the blood of immunized animals that inhibit the main processes of development of coli infection - colonization of the pathogen in the intestines of animals and the neutralization of endo- and enterotoxins. However, this vaccine lacks a shigaplike toxoid and the production technology is very complicated. It is necessary to grow individual Escherichia strains, isolate the necessary components from them, process them, then mix in a certain ratio.

A known method of obtaining a vaccine against escherichiosis of calves (RF patent for the invention No. 2070819, 1996). 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 the 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 one of the Escherichia strains after growing daily broth cultures at 37 ° C for two days and washing the microbial mass with physiological saline containing 0.3-0.4% formalin, bringing to a concentration of 50-60 billion microbial bodies in 1 ml by adding to the washout 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.

With this method of obtaining a vaccine, raw materials are obtained by culturing toxigenic Escherichia strains in Hottinger medium for 5-7 days. However, according to Pirozhkov’s data (Biological preparations for the specific prophylaxis and treatment of animal escherichiosis / M.K. Pirozhkov // Diss. Doc. Vet. Sciences - Moscow, 2002. - 298 p.) In the Hottinger broth there is a sufficiently high level of free amino acids that the synthesis of adhesion factors and toxins. The use of potassium alum, hydrogen peroxide in the vaccine can cause vaccine-related complications in vaccinated animals. Numerous technological methods used in obtaining the vaccine complicate the manufacturing technology and cost of the final product.

A known method of producing colibacteriosis toxoid (RF patent for the invention No. 2270857, 2006), which provides for the cultivation of Escherichia coli for 24 hours on a synthetic nutrient medium using citric acid, sodium chloride, magnesium sulfate, potassium phosphate disubstituted, iron sulfate, asparagine, glucose and glycerin. 0.6% formalin is added to the resulting biomass to detoxify E. coli toxins and is separated by filtration. Then sorption of inactivated colibacteriosis toxoid is carried out on an alumina hydrate gel.

A known method of producing Escherichia toxoid (RF patent for the invention No. 2213575, 2003, prototype), which consists in maintaining one-day meat-peptone broth cultures of E. coli serotypes 0141: K88ac; 0138: K88ac; 0139: K88as in the presence of 0.4% formalin at a temperature of 37-38 ° C for 25 days and passing through a Salnikov filter.

However, the authors do not report which particular toxins or toxin produced the epizootic strains of Escherichia coli selected by them, and therefore, the use of this drug is limited to any one farm.

The technical result of the invention is to increase the effectiveness of specific prophylaxis in relation to the main pathogenicity factors of Escherichia coli - thermolabile, thermostable and shigapodobny toxins and the expansion of the use of a specific drug.

This object is achieved in that the method of producing Escherichia toxoid involves the cultivation of epizootic strains of Escherichia coli in nutrient broth, inactivation of cultures by adding formalin to a concentration of not more than 0.4%, sterilizing filtration, packaging and corking. Moreover, it is of key importance that Escherichia coli strains with the genes of thermolabile, thermostable and shigatoid toxins are selected. Each strain is separately cultivated at a temperature of 37 ° C for 6-7 days with daily double stirring, then the cultures are inactivated with formalin at a temperature of 37 ° C for 14 days with daily double stirring, after which the cultures are selected and mixed in equal proportions, separated the bacterial mass using sterilizing filtration and get a complex preparation containing 3 types of toxins.

The novelty of the proposed proposal lies in the fact that the thermolabile, thermostable and shigapodobny toxins obtained in the process of cultivation of toxigenic Escherichia coli are used as antigen.

The method provides a harmless, highly immunogenic and more specific vaccine to protect animals from colibacteriosis.

In the patent and scientific literature not found similar to the claimed combination of features, allowing to obtain a technical result that was not previously achieved by known means, which allows to judge the inventive step and the novelty of the proposed proposal.

An example of a specific implementation of the method of producing Escherichia toxoid.

To obtain the Escherichia toxoid, initially among the epizootic strains of Escherichia coli, we selected those that possess the genes of thermolabile, thermostable, and shigapodobny toxins. To do this, we used AmpliSens E. coli-tox test systems (Central Research Institute of Epidemiology of the Ministry of Health of the Russian Federation, Moscow). Confirmation of toxin formation on an artificial nutrient medium was carried out using a biotest on infusoria-stylonychia (US Pat. RF 2262529, 2005). In the presence of exotoxins in the culture medium, the number of dead ciliates should be at least 70-80%, while the number of dead ciliates in a control sample containing no toxin should not exceed 5%.

The selected strains were individually inoculated into test tubes with 10 ml of nutrient broth for the accumulation of toxin Escherichia coli, which includes acid blood hydrolyzate (9-11%), baker's yeast autolysate (9-11%), peptone (0.9-1, 1%), narya chloride (0.4-0.6%), disubstituted sodium phosphate (0.05-0.15%) and potassium chloride (0.01-0.03%) (US Pat. No. 2342425, 2008), after which they were placed in a thermostat and cultured at 37 ° C for 6-8 hours until a slight turbidity appeared, indicating the growth of microorganisms. Then, the resulting cultures were transferred to flasks with 200-300 ml of a similar nutrient broth and incubated at 37 ° C for: producing thermolabile and thermostable - 6 days, and producing shigapodobny toxin - 7 days. The flasks were shaken daily 2 times a day. Before the end of the incubation, 10 ml of culture fluid was taken from each flask, centrifuged at 10 thousand rpm for 30 minutes, and determined by biotesting on the ciliates of the presence of toxins.

In the presence of toxins, formalin was added to the remaining cultures to a concentration of 0.3-0.4%. Inactivation of cultures was carried out for 14 days at a temperature of 37 ° C with daily twofold stirring. After completion of inactivation, equal volumes of cultures were combined, thus obtaining a complex preparation containing 3 types of antigens. Using sterilizing filtration, the microbial mass was separated from the culture medium. The finished preparation under aseptic conditions was packaged in sterile vials and sealed. The resulting Escherichia toxoid was tested for sterility, harmlessness and protective activity.

During the control seeding of toxoid on MPA, BCH, medium Kitt-Tarozzi, Saburo, Endo, the growth of bacterial and fungal flora was absent, which indicated the sterility of the drug.

With the intraperitoneal administration of toxoid at a dose of 0.3 ml to white mice weighing 20-22 g, oppression and death within 10 days were not noted, which is an indicator of the safety of the drug.

The test of the protective properties of Escherichia toxoid was carried out on white mice weighing 20-22 g. For this, the animals were injected with the toxoid subcutaneously twice with an interval of 7 days at a dose of 0.1 + 0.1 ml. Infection of experimental mice was performed 7 days after the last administration of toxoid. For infection, a filtrate of broth cultures of toxigenic E. coli was used, producing thermolabile, thermostable and shigapodobny toxins in a dose of 2 LD ₅₀ . Intact (unvaccinated) mice and mice twice immunized with the Kolya-Vak vaccine were used as a control.

The test results of the protective properties of Escherichia toxoid are shown in the table.

Table The effectiveness of Escherichia toxoid in experimental infection of white mice Group Dose, ml Number of animals Infecting Dose, ml results fell survived Experienced, immunized with Escherichia toxoid 0.1 + 0.1 10 0.05 0 10 Control immunized with the Kolya-Vak vaccine 0.1 + 0.1 10 0.05 four 6 Control, not immunized - 10 0.05 10 0

The table shows that after immunization of animals with the drug obtained by the present method, in 100% of cases they form a specific immunity to the pathogenic effect of E. coli toxins, while only 60% of animals survived after immunization of mice with the Kolya-Vak vaccine, and all non-immunized animals died. Therefore, the Escherichia toxoid has a pronounced protective effect against E. coli toxins and can be used as a specific biological product for the prevention of colibacillosis.

Claims (1)

A method of producing an Escherichia toxoid, including culturing epizootic strains of Escherichia coli in a nutrient broth, inactivating cultures by adding formalin to a concentration of not more than 0.4%, sterilizing filtration, packaging and capping, characterized in that they select strains of Escherichia coli that have thermostable, thermostable genes and shigate-like toxins, each strain is separately cultured at a temperature of 37 ° C for 6-7 days with daily double stirring, then inactivation of cultures of pho rimalin at a temperature of 37 ° C for 14 days with daily double stirring, after which the cultures are selected and mixed in equal proportions, the bacterial mass is separated using sterilizing filtration and a complex preparation is obtained.