RU2377013C1 - Method for production of staphylococcic anatoxin-vaccine - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: invention is related to the field of microbiology and biotechnology. Staphylococcic anatoxin-vaccine is prodyuced by detoxication of staphylococcic exo- and endotoxins by to detoxifiers - 0.2% of formalin solution for 7-9 days at 40-45°C, and then 0.6±0.1% solution of etonium for 7-9 days at 40-45°C. At the same time complete detoxication of toxins and inactivation of their allergenic properties are performed at practically 100 % of formalin consumption and etonium. Then sorption of staphylococcic anatoxin-vaccine is carried out per 1-3 mg/ml of aluminium hydroxide, and concentration of inactivated toxin-allergens is doubled by decantation of 50±5% of supernatant.

EFFECT: invention is used for faster healing of wounds, treatment of dermatitis, pneumonia of calves, piglets, prophylaxis of birds staphylococcus.

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Description

The invention relates to microbiology, in particular to a technology for the manufacture of biological products for the specific prophylaxis and treatment of animal diseases of staphylococcal etiology.

Known methods for producing inactivated viral and bacterial vaccines, toxoids are based on the use of 0.8 ± 0.2% formalin solution, UV rays, ionizing rays, etc. (Shapiro Y. S. “Microorganisms - viruses, bacteria, fungi.” Publishing House “ELBI” ". - St. Petersburg, 2003, p.156-160).

The disadvantage of these methods is the detoxification of staphylococcal exo- and endotoxins using 0.8 ± 0.2% formalin solution, which is carcinogenic and practically does not detoxify superenterotoxins and, accordingly, does not turn them into toxoids (Korotyaev A.I., Babichev S. A. “Medical Microbiology, Immunology and Virology.” St. Petersburg, Special Literature Publishing House, 1998, p. 333).

The search for more effective and harmless detoxifiers using fatty acids, hydrogen peroxide, antibiotics, photooxidation, ninhydride, tyrosine, P-propylactone was random and not recognized.

At the same time, in Japan, the production of tetanus, staphylococcal and streptococcal toxoids is based on the use of glucuronic acid, gluconate and sodium gluconate as a detoxifier (patent No. 23345 with priority dated December 6, 1961).

Moreover, with respect to staphylococcal superenterotoxins, the detoxifying effect of these inactivators (detoxifiers) is not indicated, although they are the strongest food poisons and are resistant to formalin and temperature.

It should be noted that the term superantigens (SAG) is used to define globular protein molecules produced by pathogenic microorganisms that have pyrogenic (erythrogenic) and mitogenic effects, which can cause arthritis, skin lesions, AIDS, enhance pathogenesis, etc.

The prototype is taken as “A method for producing a staphylococcal toxoid vaccine” (RU 2139091, 10.10.1999), including the cultivation of staphylococci, their sterilization, stage-by-stage detoxification with formalin: addition of formalin initially to a concentration of 0.3-0.4% with incubation at 37 -40 ° C for 7-10 days, and then to a final concentration of 0.5-0.8%, followed by incubation at a temperature of 44-46 ° C for 5-8 days, sorption on aluminum hydroxide to a concentration of 1 mg / ml and autoclaving sterilization.

The disadvantage of this method is the detoxification of staphylococcal toxins with one detoxifier - formalin, which does not ensure the complete inactivation of all types of toxins, and therefore can not turn superenterotoxins into toxoids.

A comparative assessment of the above detoxifiers and testing of new ones revealed the effectiveness of a number of bis-quaternary ammonium compounds, in particular ethonia, which was taken as the basis for the preparation of staphylococcal toxoid vaccine.

The aim of the invention is to increase the effectiveness and safety of the toxoid vaccine by detoxifying staphylococcal toxins with two detoxifiers in two stages - initially with 0.2% formalin solution, instead of 0.8% for 9-10 days, and then 0.6 ± 0.1 % ethonium solution for 7-9 days at 40-45 ° C.

The goal is achieved by reducing the concentration of carcinogenic and ineffective against staphylococcal superenterotoxins formalin from 0.8 ± 0.2% to 0.2% and the subsequent addition of 0.6 ± 0.1% etonium for complete and irreversible detoxification of all types of toxins, sorption on aluminum hydroxide at a rate of 1-3 mg / ml and decantation of 50% of the supernatant.

In this case, the initial concentration of formalin decreases from 0.2% to 0.1% due to the binding of toxins to protein molecules, and the safety of etonium (1,2-ethylene-bis- (N-dimethylcarbdecyloxymethyl) ammonium dichloride-bis-quaternary ammonium compound) is proved long-term use of it in the form of a 0.5-1% solution, ointments, etc. in humane medicine in dentistry, ophthalmology, dermatology, etc.

In the patent and scientific and technical literature, no technical solutions are found that are similar to those claimed using etonia.

The use of a 0.6 ± 0.1% solution of etonium with a decrease in the concentration of formalin from 0.8 ± 0.2% to 0.2% in the proposed method allows to achieve complete and irreversible detoxification of staphylococcal exo-, endo- and superenterotoxins and obtain a harmless and effective staphylococcal toxoid vaccine.

The established possibility of complete and irreversible detoxification of a complex of staphylococcal exo-, endo-, and superenterotoxins by using at the first stage 0.2% formalin solution instead of 0.8%, and in the second stage 0.6 ± 0.1% ethonium, sorption on aluminum hydroxide Decantation of 50% of the supernatant allowed us to develop an optimal process scheme for obtaining staphylococcal toxoid vaccine in an industrial environment.

The alleged invention is illustrated by the following examples.

Example 1

A method of obtaining a staphylococcal toxoid vaccine

Grown staphylococci with exo- and endotoxins on a liquid synthetic medium in 2-liter bio-bottles with a volume of medium equal to 1 liter, for 12 ± 2 days up to 10.0 ± 2.0 billion / cm ^{3 of a} suspension of microorganisms were autoclaved at 1.0 atm for 30 minutes, then a detoxification of the toxin complex was carried out initially with a 0.2% formalin solution for 9-10 days at 40-45 ° C, and then with a 0.6 ± 0.1% ethonium solution at 40-45 ° C in within 7-9 days. After detoxification, sorption was performed on 1-3 mg / ml aluminum hydroxide and the concentration of inactivated toxins with microorganisms was increased by decantation of 50 ± 5% of the supernatant.

The resulting toxoid vaccine was packaged in 20, 100 and 200 ml vials and sterilized at 1.0 atm for 20 minutes.

Example 2

Staphylococcal Toxoid Vaccine (CAB) Safety Assessment

A study of the toxicity of CAB was performed on 12 white mice weighing 20 ± 2.0 g by intraperitoneal (6 gol) and subcutaneous (6 gol.) Administration of 1.0 ml of the biological product.

All animals without visible necrotic, inflammatory processes and general toxicosis remained alive when observed for 15 days.

Example 3

Dog harmlessness test

For research, 6 outbred puppies at the age of 2-3 months weighing 3-4 kg were used. In experimental animals, 5 ml of CAB was injected simultaneously subcutaneously and intramuscularly. For 15 days, all animals remained alive, and at the injection site of the biological product, no inflammatory and purulent-necrotic lesions were noted.

Example 4

Comparative evaluation of the effectiveness of treatment of burn wounds in pigs

A skin burn in piglets in the amount of 7-10 cm was obtained by applying for 5-7 minutes several layers of gauze moistened with 5% carbolic acid solution - 18 goals.

The treatment was carried out in the form of lotions twice a day using 2 variants of the staphylococcal toxoid vaccine obtained using detoxification of toxins with 0.8% formalin solution (1st control variant) and 0.2% formalin solution with 0.6% ethonium solution (test option). It was found that the granulation tissue in piglets treated with the test variant of the toxoid vaccine appeared 2-3 days earlier than the control, granulation of the entire wound surface occurred by 20-22 days of treatment, and in the treatment of burn wounds with the control vaccine by 25- 27 day. Therefore, in the treatment of burn wounds in pigs with the staphylococcal toxoid vaccine obtained using ethonia, the wound healing process was completed 3-5 days earlier than when using formol vaccine.

Example 5

Comparative efficacy of staphylococcal toxoid vaccine with etonium

The research used 45 cows with catarrhal and purulent-catarrhal mastitis (from gr. Mastos - breast, nipple and fr. Caftare - inflammation of the mucous membrane). During bacteriological examination of milk from sick cows, Staphylococcus aureus cultures, S. aureus, were isolated.

Sick animals were divided into three groups of 15 cows.

In the first control experiment, the formolanatoxin vaccine was used, in the second mastisan-A, containing in the suspension sunflower oil, penicillin, streptomycin, norsulfazole and wax. Before the administration of drugs, milk was removed from the udder, the nipple was rubbed with 70% alcohol. Mastisan-A was administered intracisternally daily in a dose of 15 ml, and formolvaccine and staphylococcal toxoid vaccine with ethonium 4-5 ml. It was found that in the treatment of cows with mastitis, CAB with ethony recovered in 6-7 days, 13 cows and 2 cows recovered after 9 days, with CAB with formalin, treatment success was achieved only after 9-10 days, and with using mastisan after 11-12 days. A significant drawback in the treatment of cows with mastisan-A is the presence in the preparation of antibiotics that are excreted in milk during treatment and after the drug is discontinued.

Claims (1)

A method of producing a staphylococcal toxoid vaccine, comprising phased detoxification of tuberculous exo-, endo- and superenterotoxins in suspension with autoclaved staphylococci, sorption of toxoid on aluminum hydroxide, sterilization, characterized in that the detoxification of staphylococcal toxins is carried out initially with two 0.2% detoxifier formalin solution for 7-9 days at 40-45 ° C, and then a 0.6 ± 0.1% ethonium solution for 7-9 days at 40-45 ° C, while the concentration of inactivated toxins with microorganisms is increased double the decantation of 50 ± 5% of the supernatant.