RU2412992C1 - Strain of bacterium escherichia coli eb387 for protection of animals of canine family against toxicoses induced by cytotonic toxins of a1b5, type and based on it probiotic medication - Google Patents

Abstract

FIELD: medicine. ^ SUBSTANCE: strain is obtained by insertion into genome of Escherichia coli, isolated from clinically healthy representative of canine family, of genes, determining synthesis of microcin C51, B-subunit of toxin, as well as vaccine version of elt-operon and A-subunit, enhancing immune response. Strain possesses expressed adhesion to mucous membrane of intestine of animals of canine family. Frozen dried culture of Escherichia coli EB387 represents probiotic medication for protection of animals of canine family against toxicoses, induced by cytotonic toxins of A1B5 type. ^ EFFECT: normalisation and stabilisation of qualitative and quantitative composition of gastrointestinal tract microflora in representatives of canine family. ^ 2 cl, 3 ex

Description

The invention relates to veterinary medicine and biotechnology and can be used to protect domestic and wild representatives of the canine family (Canidae) from toxicosis caused by cytotonic toxins of type A ₁ B ₅ .

Cytotonic toxins of type A ₁ B ₅ are widely distributed among various taxonomic groups of microorganisms: enterobacteria, vibrios, pseudo- and aeromonads, helicobacteria. In recent years, there has been an expansion in the circle of bacteria producing these toxins, which is associated with the drift of the genes encoding them in bacterial populations.

Toxins of this group affect the digestive and excretory systems of the body, reproductive organs, and skin integuments. Activate intracellular parasites, aggravate the course of viral and parasitic diseases. These toxins can also affect people, being one of the main causes of travelers' diarrhea.

Features of the formation of antitoxic immunity make therapeutic and prophylactic drugs based on parenteral administration ineffective.

The closest in essence and the achieved positive effect to the claimed strain are strains of Escherichia coli M17 (RF patent No. 1633817) and E. coli BKM SK-322D (RF patent No. 1819428), which have antibacterial activity and are intended for the prevention of intestinal diseases of farm animals, but lacking adhesion to the cells of the mucous membrane of the gastrointestinal tract and the E. coli strain of the RLMB (RF patent No. 2259213), which has adhesive properties, but is not able to cause an effective local immune response due to the lack of recombinant plasmid pLTB gene fragment of toxin A subunit conferring gene product superantigen properties.

The objective of the invention is to obtain a new probiotic strain with pronounced adhesion to the intestinal mucosa of carnivores of the canine family and is able to protect them from toxicosis caused by toxins of type A ₁ B ₅ due to the formation of local immunity due to class A immunoglobulins and the screening of specific cellular receptors for these toxins - gangliosides GM1.

The task is achieved by the fact that genes determining the synthesis of C51 microcin and the vaccine variant of the elt toxoid with a deletion of the toxin effector A subunit in the gene providing loss of toxic function were integrated into the genome of the E. coli strain isolated from a clinically healthy member of the canine family. At the same time, the presence of the A subunit sharply enhances the immune response due to the superantigenic properties of the latter. The presence of a receptor B-subunit in the genome of a strain provides shielding of cellular receptors from the action of cytotonic toxins of type A ₁ B ₅ . Microcin C51 inhibits the development of pathogenic and conditionally pathogenic enterobacteria, while not exerting a depressing effect on normoflora. In addition, the strain Escherichia coli EB387 has a stimulating effect on the obligate microflora of the gastrointestinal tract, in particular, contributing to the restoration of its species and quantitative composition in dysbiotic conditions.

Strain E. coli No. 97/3 was isolated from the feces of a clinically healthy dog. Primary identification is based on morphological and cultural properties. Finally, the species affiliation was determined on the basis of the AP test. The strain did not contain plasmid DNA and did not bear the determinant of drug resistance, but had pronounced adhesive properties with respect to guinea pig erythrocytes.

Recombinant plasmid p ΔABLT was obtained on the basis of the natural plasmid pH74114, which contains a full-sized elt operon.

For this, plasmid DNA was isolated and purified from E. coli strain H74114. Isolation and purification was carried out as follows. Bacterial cells from 10 ml of overnight broth culture were besieged by centrifugation and resuspended in 0.5 ml of STET buffer of the following composition: sucrose 8%, Triton X-100 - 0.5%, EDTA 25 mM, Tris-HCL 25 mM, pH 8.0 . After that, 0.25 ml of 0.3 M NaOH was added to them, placed in a water bath at a temperature of 70 ° C and incubated for 15 minutes. Then, a mixture of equal volumes of phenol with chloroform was added to the sample, and after vigorous stirring, it was centrifuged at 14000 rpm for 10 minutes. The aqueous phase was taken, to which 0.07 ml of a 3M sodium acetate solution and 0.7 ml of isopropanol were successively added, mixed and centrifuged. The DNA precipitate was washed twice with 70% ethanol, dried under vacuum and dissolved in 0.05 ml of deionized water.

The plasmid DNA preparation pH74114 thus obtained was hydrolyzed by PstI restriction enzyme. The reaction was carried out in a buffer containing 10 mM Tris-Hcl (pH 8.5 at 37 ° C), 10 mM MgCl, 100 mM KCl at 37 ° C for 1 hour. The reaction was stopped by heating (15 min at 65 ° C). The DNA of the vector plasmid was treated in the same way, the restriction was mixed at a ratio of 1: 4 (vector: insert), and the bacteriophage T4 DNA ligase was treated at 4 ° C overnight.

The obtained ligase mixture was transformed cells of a laboratory strain of E. coli DH5. The transformation was carried out as follows: the overnight culture of the recipient strain was diluted 100 times with LB medium and grown to the middle of the logarithmic growth stage. Lets in a volume of 40 ml were precipitated by centrifugation and washed with 0.15 M NaCl, suspended in 2 ml of a 50 mM CaCl ₂ solution. After 20 minutes of incubation, 20 μl of the ligase mixture was added to them and incubated for another 40 minutes in an ice bath. After temperature shock (42 ° C, 3 minutes), the cells were diluted with 0.5 ml of LB medium, incubated at 37 ° C for 90 minutes, plated on plates with agarized LB medium and placed in a thermostat at 37 ° C. The grown colonies were selected and analyzed for the presence of the elt operon. According to the results of the analysis of clones, one was selected that carried a full-sized elt-operon. Plasmid DNA was isolated from the cells of this clone by the above method. The isolated DNA was sequentially treated with restriction endonucleases XbaI, BclI and nuclease S ₁ , extracted with neutral phenol and reprecipitated with 2 volumes of ethanol. The preparation thus obtained was treated with T4 bacteriophage DNA ligase and, through transformation, introduced into E. coil strain No. 97/3. As a result, a clone designated as E. coli 97/3/28, which contained the recombinant plasmid p ΔABLT, was thus constructed and selected.

Plasmid p74, which determines the synthesis of C51 microcin, was isolated from the cells of E. coli M17 strain by the above method. The resulting DNA was transformed with strain E. coli 97/3/28, which contained the recombinant plasmid p ΔABLT. The transformation efficiency was 10 ² transformants per 1 μg of DNA. Electrophoretic analysis of plasmid transformant profiles showed the presence of two plasmids p ΔABLT and p74. Thus, a clone designated as E. coli EB387 was constructed and selected, which contained the recombinant plasmid p ΔABLT and plasmid p74.

To determine the immunological activity of the protein synthesized under the control of plasmid p ΔABLT, the method of double radial diffusion according to the Uhterloni method was used. For this, E. coli strain EB387 cells were cultured in 250 ml of BCH medium for 24 hours, precipitated by centrifugation, and resuspended in 0.01 M Tris-Hcl buffer pH 8.6 containing 0.15 NaCl. The resulting cell suspension was processed on an ME 150 ultrasonic disintegrator.

For the reaction, 1% agar prepared on the above buffer and polyclonal serum to the full-length LT molecule were used.

The resulting lysate formed precipitation bands with polyclonal serum to a full-length LT molecule at a dilution of 1: 128. The immunoblot delivered with this lysate showed the presence of two bands with a molecular weight of 24 and 12.7 kD, respectively, which corresponds to the molecular weights of A and B of the LT subunits.

In order to study the pathogenicity of the probiotic strain E. coli EB387, a broth culture of the strain at a dose of 1-5 × 10 ⁹ was administered orally and intraperitoneally to laboratory mice weighing 16-20 g. In no case of animal death was observed. At autopsy, pathological changes in the organs were not observed.

When the drug was administered orally at a dose of 1-5 × 10 ⁹ on the head of puppies of a dog, veil of a polar fox and silver-black fox, as well as young rabbits, toxicity was not detected. The term of the persistence of the probiotic strain in the body of canine representatives with oral administration of at least 280 days (observation period).

The resulting bacterial strain E. coli EB387 was deposited in the collection of strains of pathogens of fur-bearing animals of the Museum of Bacterial and Viral Cultures of the Scientific Research Institute of Fur Breeding and Rabbit Breeding named after V.A. Afanasyev RAAS (NIIPZK).

Characterization of E. coli strain EB387.

The recombinant resident antitoxic strain E. coli EB387 has a prolonged effect, the microcin C51 produced by it inhibits the development of toxigenic enterobacteria, and the deleted variant of the thermolabile toxin shields specific receptors on the surface of enterocytes from the action of holatoxin and activates specific immunity. The resident properties of the strain ensure its inclusion in the composition of the microbiota, which allows you to try it less often and in smaller doses.

Genetic markers: Contains genes for microcinogenesis (C51), the deleted vaccine variant of the Escherichia elt operon, is resistant to kanamycin (100 μg / ml) and tetracycline (50 μg / ml).

Morphological, cultural, enzymatic properties: gram-negative, non-spore-forming, motile rods with rounded ends of size (1.2-1.5) × (2.0-5.0) microns. They grow well on MPA, Hottinger agar medium, minimal media with glucose and glycerin. Daily cultures on liquid media form uniform turbidity. On dense media, gray-yellow S-form colonies are formed, on Endo medium red colonies with a metallic luster (1-2 days at 37 ° C).

Optional anaerobic. Temperature optimum 37 ° C.

It breaks down glucose, lactose, sucrose, maltose, rhamnose, arabinose, beckoning, inositol to form acid and gas. It does not utilize sorbitol, sodium citrate and malonate. Urease activity is absent. Hydrogen sulfide does not form. Synthesizes galactosidase. Indole forms. Does not synthesize phenylalanine deaminase. There is no gelatinase activity, milk ferments. The activity of lysine decarboxylase and ornithine decarboxylase is detected, arginine dehydrolase is absent.

Animal Virulence (LD ₅₀ ): Not pathogenic for white mice, rats, rabbits, dogs, arctic foxes and foxes when taken orally.

Biological activity in the sensitive system: on Adams and M9 media, it inhibits the indicator strain E. coli B with a growth suppression zone from 10 to 50 mm, reacts positively with anti-LT polyclonal serum in the Uhterloni double diffuse precipitation reaction. Hemmagglutination of guinea pig erythrocytes reveals the adhesive antigen of E. coli strain EB387.

Shelf life and frequency of passages on a sensitive system: reseeding on MPA every two months, checking for specific activity every six months. In a lyophilized form, the strain is stored for at least 1 year at a temperature of 4 ° C.

The state of the strain is monitored in two ways: once every three months, the cultural, morphological and biochemical properties are checked and, before use, they confirm their specific activity by testing for the presence of microcinogeny on Adams medium using E. coli B indicator culture and detecting toxoid biosynthesis in an immunochemical test with monoclonal antibodies.

Based on the obtained E. coli strain EB387, a probiotic preparation was created, which was a lyophilized culture of microorganisms intended for the prevention of toxicosis in canine animals caused by cytotonic toxins of type A ₁ B ₅ .

The protective activity of the probiotic preparation based on E. coli strain EB387 was tested on representatives of the canine family — veil foxes on the basis of the Pushkinsky and Saltykovsky breeding plants in the Moscow Region. It was found that the drug has a long term persistence in the intestines of representatives of the canine family, taking the drug contributed to the normalization of intestinal microflora and prevented the development of toxicosis in experimental animals of the canine family.

Example 1. Terms of persistence and elimination of the drug.

In experiments on foxes of the veil breed at the Pushkin and Saltykovsky breeding plants, it was found that after a weekly course of taking a probiotic at a dose of 1.5 million CFU per head of the experimental animal, the probiotic strain E. coli EB387 was seeded as a part of the luminal microflora in within 260 days (observation period).

In experiments performed on female veal foxes, it was found that after their treatment with a probiotic preparation based on E. coil EB387 strain, the latter was found in the intestinal microflora of the puppies born to them during preparation for the gon.

Example 2. The effect of the drug on the intestinal microflora.

In experiments conducted on 30 heads of young foxes of the veil breed at the Saltykovsky breeding plant, it was found that after a weekly course of taking a probiotic at a dose of 1.5 million CFU per head of the experimental animal, the latter normalized the qualitative and quantitative composition of intestinal microflora: they disappeared conditionally pathogenic non-fermentative Escherichia, the number of normal Escherichia coli increased by 1-2 orders of magnitude and reached normal values of 10 ⁷ ; the number of lactobacilli and enterococci also increased by 2-3 orders of magnitude to a level of 10 ⁸ -10 ⁹ . The number of bifidobacteria did not change and corresponded to the norm - 10 ⁹ -10 ¹¹ . Thus, under the influence of the drug, normalization and stabilization of the qualitative and quantitative composition of the normoflora of the gastrointestinal tract of the canine family occurs.

Example 3. The protective activity of the drug.

In experiments on laboratory animals, treatment of white mice with a probiotic preparation based on E. coli strain EB387 prevented the onset of diarrhea syndrome and death of the latter when they were infected with a pathogenic strain of Escherichia coli. The treatment of females and young veil arctic foxes on the basis of E. coli EB387 strain prevented their pre-registration death and increased the yield of puppies by 0.6 heads per female compared to the untreated control.

Claims (2)

1. The bacterial strain Escherichia coli EB 387, deposited in the collection of pathogen strains of fur-bearing animals and rabbits of the Museum of Bacterial Culture, GNU NIIPZK im. VAAfanasyeva RAAS No. EB 387, designed to protect animals of the canine family from toxicosis caused by cytotonic toxins of type A ₁ B _5. 2. A probiotic preparation for the protection of canine animals from toxicosis caused by cytotonic toxins of type A ₁ B ₅ , characterized in that it is a lyophilized dried culture of Escherichia coli according to claim 1.