RU2372937C1 - Method of production of colibacillosis toxoid-vaccine - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: invention refers to microbiology and biotechnology, to vaccine production for prevention of infectious diseases in particular. Method includes cultivation of Escherichia using culture medium, detoxication of exo- and endotoxins in suspension with autoclaved Escherichia performed using formalin solution, sorption using aluminium hydrate. At that for Escherichia cultivation synthetic medium is used. Toxins detoxication is performed in sequence using two detoxifiers - initially 0.2% formalin solution at 40±1°C during 6-7 days and then 0.5±0.1% ethonium solution at 40±1°C during 7-9 days with further sterilisation at 1.0 atm during 20 minutes.

EFFECT: produced toxoid-vaccine is characterised by high activity and is used for prevention of animal infectious diseases.

4 ex

Description

The invention relates to microbiology and biotechnology, in particular to the manufacture of biological products for the specific prevention of colibacillosis of calves, piglets and birds.

Known methods for producing formol vaccines and toxoids are based on the detoxification of toxins and the inactivation of viruses and bacteria by 0.8 ± 0.2% formalin solution or its derivatives (Vorobyov A.A. “Anatoxins." M., Medicine, 1965, pp. 137-355 )

The colibacteriotic vaccine is based on the cultivation of Escherichia on Hottinger broth, sterilization, detoxification of toxins with a 1% formalin solution, sorption on aluminum hydroxide with the addition of merthiolate as a preservative.

Due to the carcinogenic properties of formalin, merthiolate, and the incomplete detoxification of many supertoxins, some researchers tested β-propylactone, tyrosine, hydrogen peroxide, antibiotics, and photooxidation as detoxifiers. Particular attention is paid to the replacement of meat hydrolysates, casein with synthetic nutrient media for the cultivation of microorganisms.

However, with respect to the colibacillus vaccine, no significant changes have occurred.

The prototype is a method of producing a colibacteric toxoid vaccine, including the cultivation of E. coli in Hottinger broth, sterilization and detoxification of toxins with 1% formalin solution, sorption on aluminum hydroxide, followed by the addition of a preservative - merthiolate (Osidze N.D., Borisovich Yu.F. , Kirilov L.V. Veterinary preparations. - M .: Kolos, 1981, p.219-225).

The disadvantage of this method is the cultivation of Escherichia on the Hottinger hydrolyzate and detoxification of 0.8 ± 0.2% formalin solution.

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 (1,2-ethylene-bis- (N-dimethylcarbdecyloxymethyl) ammonium dichloride). The safety and effectiveness of etonium is confirmed in its use in humane medicine as ointments, pastes and solutions for dermatitis, a crack in the nipples, rectum, for wound healing, for keratitis and stomatitis, etc.

The aim of the invention is to provide complete and irreversible detoxification of a complex of colibacteriosis toxins after growing Escherichia in a synthetic medium free of meat and casein ballast substances, reducing the formalin concentration to 0.2% by introducing a second ethonium detoxifier at a concentration of 0.5 ± 0.1% for final detoxification at 40 ± 1 ° C for 6-7 days.

The goal is achieved by reducing the concentration of carcinogenic formalin from 0.8% to 0.2% with the subsequent introduction of 0.5 ± 0.1% etonium for complete and irreversible detoxification of all types of toxins, including enterotoxins that formalin does not detoxify, and, therefore, cannot turn them into toxoids.

In this case, the initial concentration of formalin is reduced to 0.05%, and ethonia to 0.2-0.3% due to their binding to protein molecules of toxins and microorganisms.

In the patent and scientific and technical literature, no technical solutions were found that are similar to those claimed using etonia, which indicates the novelty of the proposed solution.

The use of a 0.5 ± 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 a stable completeness of detoxification, polymerization of the whole complex of colibacteriosis toxins and obtain a harmless and effective colibacteriosis toxoid vaccines.

The established possibility of complete and irreversible detoxification of all types of colibacteriosis toxins, including enterotoxins, by using at the first stage of detoxification a 0.2% formalin solution instead of 0.8 ± 0.2% and in the second stage 0.5 ± 0.1% ethonia , possessing not only detoxifying, but preserving, bactericidal and fungicidal properties, allowed us to propose a rational technology for the manufacture of an effective and harmless colibacteric toxoid vaccine without a mercury-containing preservative - merthiolate.

The invention is illustrated by the following examples.

Example 1. Obtaining colibacillus toxoid vaccine (CAV).

Cultivated Escherichia on a synthetic medium in 2-liter biobottles with a volume of 1 l of medium for 3 days with a concentration of 60 ± 10 billion / ml of microorganisms were autoclaved at 1.0 atm for 30 minutes. The concentration of molecular toxins was 1.2 ± 0.2 mg / cm ³ .

Formalin was added to the resulting suspension of autoclaved Escherichia with soluble toxins to 0.2% concentration for detoxification at 40 ± 1 ° C for 7 days, and then ethonium was added to 0.5% concentration to complete detoxification at 41 ± 1 ° C in during 7 days of all types of colibacteriosis toxins, including enterotoxins, which formalin is not able to detoxify.

After sorption of the toxoid vaccine on aluminum hydroxide (3 mg / cm ³ ), the drug was packaged in 20.0 and 100.0 ml vials and sterilized at 1.0 atm for 20 minutes.

The resulting toxoid vaccine has been tested for safety and efficacy.

Example 2. Test colibacteriosis vaccine for harmlessness in white mice and 15-30 days of age of piglets.

The toxicity study was carried out on 8 white mice weighing 18-20 g and 10 piglets by subcutaneous injection of 0.5 cm ³ and 5.0 cm ^{3 of the} biological product.

When observed for 10 days, all animals remained clinically healthy, with no visible changes at the injection site.

Example 3. Testing colibacillus vaccine for activity.

The experiment used 16 calves, 18 goals of piglets 15-30 days of age and 10 white mice.

Vaccination of animals was carried out by subcutaneous administration of the drug to 0.3 ml of white mice, 5.0 ml of calves and piglets with an interval of 7 days. The control was 8 white mice, 5 heads of piglets and 5 heads of calves that were not vaccinated.

15 days after the second vaccination, the animals were subcutaneously injected with a suspension of virulent Escherichia in a dose of 10-20 thousand microbial bodies for white mice, and 150 thousand for piglets and calves. The specified number of Escherichia infection was carried out in control animals.

Observation of animals was carried out for 15 days. It was found that all control 8 heads of white mice became ill and died, and the calves and piglets were treated when clinical signs of the disease appeared.

Among vaccinated white mice, two mice became ill and died, and the remaining eight mice remained alive without signs of disease.

Among vaccinated calves and piglets, there were no signs of disease 15 days after infection.

Example 4. Test CAV on 8 pregnant sows and 10 pregnant cows.

In pregnant sows, the vaccine was administered subcutaneously two months before farrowing twice with an interval of 7 days, and for pregnant cows 30-45 days before calving three times at 7.0 cm ³ with an interval of 7-9 days. The animals did not have abortions; there were no necrotic lesions at the injection sites. The young calves (calves and piglets) did not have diarrhea.

Claims (1)

A method of producing a colibacteriotic vaccine, including the cultivation of Escherichia in a nutrient medium, the detoxification of exo- and endotoxins in suspension with autoclaved Escherichia with formalin solution, sorption on aluminum hydroxide, characterized in that synthetic bacteria are used for the cultivation of Escherichia, and the detoxification of toxins is carried out sequentially with two detoxifiers, initially two 0.2% formalin solution at 40 ± 1 ° C for 6-7 days, and then 0.5 ± 0.1% ethonium solution at 40 ± 1 ° C for 7-9 days, followed by sterilization minutes at 1.0 atm for 20 minutes.