RU2252031C1 - Method for production of immune serum to virulent strain bacillus anthracis antigen - Google Patents

Abstract

FIELD: medicine microbiology.

SUBSTANCE: claimed method includes administering before contamination to the animal subsequently in right pope then after 14 days in left pope 0.2 ml of non-complete Freund's adjuvant with equal volume of physiological salt solution. Subsequent administering after 14 days in right pope up to 10 LD₅₀ of Bacillus anthracis 81/1 spore dredge doesn't cause animal death for long period (monitoring time is 35 days).

EFFECT: immune serum for screening of anthrax diagnosis preparation.

1 dwg, 1 tbl, 8 ex

Description

The invention relates to medical microbiology and for the production of immune sera from animals infected with a virulent B.anthracis strain.

It is known that guinea pigs are highly sensitive to the causative agent of anthrax and die within a short time (3-4 days) after infection. Therefore, to study the dynamics of antibodies or specific antigenemia, non-virulent (single-plasmid) strains of this microorganism are usually used (Identification of specific antigens in experimental anthrax. Abalkin V.A., Sergeeva L.V., Cherkassky B.L. // Journal of microbes, biol., epidemiol. 1989, No. 12.-C.63-68).

It is possible to use cows that are infected with large doses of the pathogen (Factors of specific and nonspecific protection in cows experimentally infected with anthrax. Buravtseva N.P., Rakitina E.L., Nelyapin N.M. et al. // Current issues of immunodiagnosis. especially dangerous infections. Part 1., Stavropol, 1986).

The disadvantage of the first method is that single-plasmid strains cause the formation of antibodies to specific antigens, which does not allow to reveal the full spectrum of antibodies in animal sera. This is possible only with immunization with a virulent strain of this microorganism. The second method is expensive.

The closest analogue of the proposed method is the publication "Factors of specific and nonspecific protection in cows experimentally infected with anthrax."

The purpose of the invention is the production of immune sera from guinea pigs after administration to animals of a spore suspension of a virulent strain of B. anthracis.

This goal is achieved by the fact that before the infection, the guinea pigs take turns - first in the right groin, then after 14 days 0.2 ml of Freund's incomplete adjuvant (NAF) is injected into the left groin with an equal volume of physiological saline (experimental group).

The control group of animals injected with 0.4 ml of physiological saline.

14 days after the last administration of NAF, animals of both groups were infected by the introduction of B. anthracis 81/1 spores (5 animals per dose) in the right groin of 20, 200, 2000.

Animals of the control group infected with 200 and 2000 spores die within 3-4 days, while animals of the experimental group infected with these doses of spores remain alive for 35 days (observation period). During bacteriological examination of organs and blood on days 21 and 35, the causative agent of anthrax was not isolated.

In the gel immunodiffusion reaction (RID) with culture filters (CF) of B. anthracis 81/1 (tox ⁺ , cap ⁺ ) STI (tox ⁺ , cap ^- ) 81/1 / 1TR (tox ^- , cap ^- ) animal serum titers the experimental groups were 1: 4-1: 16.

In RNGA with antigenic diagnostics prepared using CF of B. anthracis STI, Davis (tox ^- , cap ⁺ ), 81 / 1TR, antibody titers of the sera of these animals ranged from 1: 80-1: 640, with non-sensitized red blood cells - 1:20 .

The specificity of RNGA was confirmed by RTNGA and RNat.

The proposed method for producing anthrax serum from guinea pigs infected with a virulent strain may be useful for evaluating the developed diagnostic drugs.

Examples of specific performance, confirming the implementation of the proposed method

Example 1. The introduction of animals incomplete adjuvant Freund

Equal volumes of incomplete Freund's adjuvant and 0.85% sodium chloride solution were mixed with a medical syringe and injection needle until a stable emulsion was obtained. 0.4 ml of the emulsion was administered to guinea pigs in the right groin. After 14 days, a similar emulsion was administered to these animals in the left groin.

The control group of animals was injected with 0.4 ml of 0.85% sodium chloride solution.

Example 2. Obtaining spore suspension of B. anthracis 81/1

B.anthracis 81/1 was plated on Hottinger agar, plates were incubated at 37 ° C for three days, then at room temperature for three days. The grown culture was looped off into tubes into which a 0.85% sodium chloride solution was added. The biomass was suspended using a pipette and heated at 80 ° C for 10 min, resuspended after heating, left in the refrigerator (4 ° C) for 12 hours.

The remaining suspension was aspirated and titrated with 0.5 ml in 4.5 ml of saline, 0.1 ml were sown on Hottinger agar to determine the concentration of viable spores.

Example 3. Infection of guinea pigs with spores of B. anthracis 81/1

On the day of infection of the animals, 14 days after the last administration of NAF, the spore suspension was re-weighted to determine the concentration of spores. The animals of the experimental and control groups were injected in the right groin with 20, 200, 2000 spores in a volume of 0.5 ml.

Example 4. Bacteriological examination of dead animals

Usually, guinea pigs of the control group infected with 200, 2000 spores of B. anthracis 81/1, and some animals infected with 20 spores of this microorganism died within 3-4 days.

In the experimental group, part of the guinea pigs infected with 2,000 spores died during this time, and animals infected with 20 and 200 spores of B. anthracis 81/1 remained alive for a long time (observation period 35 days).

LD _{50 of} B. anthracis 81/1 for animals of the control group was approximately 20 spores, for animals of the experimental group - 2000 spores (see table).

When examining the fallen animals, edema was noted at the site of introduction of the pathogen; at autopsy, there was blood filling with hemorrhages in the liver and spleen.

When inoculated on the Hottinger agar, imprints of the internal organs of these animals were isolated by B. anthracis, which was identified before sensitivity to phage K and in the pearl necklace test.

Example 5. Bacteriological examination of survivors after infection of guinea pigs, blood sampling in animals

On days 21 and 35, the surviving guinea pigs after chloroformation were opened. Blood was taken from the heart. Shelter and imprints of internal organs were seeded on Hottinger agar.

At autopsy on days 21 and 35 after infection, the internal organs of animals were without features. B. anthracis was not isolated from the internal organs and blood of these animals.

After checking for sterility, the blood serum of these animals was aspirated, inactivated at 56 ° C for 30 minutes. 1: 100% merthiolate was added to inactivated sera and stored in a refrigerator until use in indirect hemagglutination (RNGA) and gel immunodiffusion (RID) reactions.

Example 6. Obtaining antigenic red blood cell diagnostics

Optimal dilutions of culture filtrates (CF) of various B. anthracis genotypes were used for sensitization of formalized tannizated sheep erythrocytes.

Cultural filtrates of B. anthracis STI, 81 / 1TR, Davis were obtained according to the previously described method (Barkov A.M., Lipnitsky A.V., Evteeva E.V. Isolation and antigenic characteristics of the components of the cultural filtrate Bacillus anthracis. // Biotechnology, 2000 No. 6.-C.27-33).

Erythrocyte sensitization was carried out according to (Bushanan T.M., Feeley J.C., Haues P.S., Brachman P.S. "Anthrax inderect hemagglutination test // J. Immmunol, 1971.-Vol.107.-P.2631-2636").

Example 7. Verification in RNGA of the activity and specificity of the serum of guinea pigs that survived after infection with B. anthracis 81/1.

RNGA was set according to the standard method in a volume of 0.025 ml. As diluent used 1% normal rabbit serum in saline pH 7.2. Test sera were titrated in a diluent from 1:10 to 1: 1280. After making diagnosticum in a volume of 0.025 ml, the plates were shaken, incubated at 37 ° C for 40 min. The result was taken into account for 20 minutes.

The specificity of RNGA was confirmed by the controls: diluent + erythrocyte diagnosticum; serum of the investigated guinea pigs + non-sensitized tanized red blood cells.

Serum titers obtained on days 21 and 35 after infection with B. anthracis 81/1 spores were made for CF-based diagnosticum. B. anthracis STI 1: 320-1: 640; for diagnostic kits based on CF B. anthracis 81 / 1TR and Davis-1: 80-1: 320 (see table).

The specificity of RNGA is also confirmed by RTNGA and RNAT.

Example 8. The activity of the serum of guinea pigs surviving after infection with B. anthracis 81/1 in RID

Guinea pig sera were not equally active in RID with CFs of various B. anthracis genotypes and formed precipitate zones in the division 1: 4-1: 16. (see drawing).

Thus, the preliminary introduction of NAF to guinea pigs leads to the long-term survival of animals after infection with 10 LD _{50 of} B. anthracis 81/1 with the formation of antibodies to various antigens of the virulent B. anthracis strain.

The use of the sera of such animals will be useful for (selection) evaluation of anthrax diagnostic drugs.

Claims (1)

A method of obtaining immune serum to antigens of the virulent strain of Bacillus anthracis, characterized in that the guinea pigs are alternately before infection, first in the right, then 14 days later they inject 0.2 ml of incomplete Freund's adjuvant with the same volume of physiological saline into the left groin, 14 days after of the last injection of Freund's incomplete adjuvant, up to 10 LD _{50 of the} spore suspension of Bacillus anthracis 81/1 are introduced into the right groin of guinea pigs, all infected animals survive up to 35 days (observation period).