RU2347806C2 - Fusobacterium necrophorum subspecies necrophorum bacteria strain for making diagnostic and preventive preparations against animal necrobacteriosis - Google Patents

Abstract

FIELD: chemistry; biochemistry.

SUBSTANCE: present invention pertains to microbiology. Obtained is a strain of the Fusobacterium necrophorum subspecies necrophorum bacteria, deposited in Research Institute "ККМ ГНЦ ВБ" "Vector" B-1076.

EFFECT: obtaining a strain, which can be used for making diagnostic and preventive preparations against animal necrobacteriosis.

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Description

The invention relates to biotechnology and agriculture, in particular to a strain of bacteria Fusobacterium necrophorum subspecies necrophorum deposited at the Research Institute of KKM SSC VB "Vector" under the number B-1076 for the manufacture of diagnostic and preventive drugs against animal necrobacteriosis.

A number of bacterial strains of the species Fusobacterium necrophorum are known that are the causative agents of necrobacteriosis in farm animals and humans: Fusobacterium necrophorum subspecies necrophorum strain FnSI (CAA48747, NCIBI), as well as Fusobacterium nucleatum subspecies vincentii strain ZPCC1bferumfentus Jumphocumferbum fungus and cellulose cellulose fungi

The closest technical solution (prototype) is the ATCC 28256 strain (AAO 12762, NCIBI) of the bacterium Fusobacterium necrophorum subspecies necrophorum, used to obtain diagnostic and prophylactic drugs against pathogens of cattle necrobacteriosis. However, a number of nucleotide sequences of gene fragments were not determined for this strain: the coat protein (rpoB), DNA gyrase (DNA gyrase B protein gene, TopoisomerasaII), hemagglutinin (ompH).

An object of the present invention is to expand the arsenal of bacterial strains of Fusobacterium necrophorum subspecies necrophorum, which can be used for the manufacture of diagnostic and prophylactic drugs against animal necrobacteriosis. Based on the claimed strain, it is possible to differentiate the isolated isolates from other anaerobes morphologically similar to Fusobacterium necrophorum, to differentiate into subspecies: subspecies necrophorum and subspecies funduliforme, as well as to select industrial crops for the manufacture of vaccines.

The problem is solved by a new strain of bacteria Fusobacterium necrophorum subspecies necrophorum, deposited at the Vector Scientific Research Institute of KKM SSC "Vector" under the number V-1076 for the manufacture of diagnostic and prophylactic drugs against animal necrobacillosis.

The strain deposited in the Research Institute of KKM SSC VB "Vector" under the number V-1076 was allocated on December 2, 1995 from a sick first-calf in the Vysokogorsky district of the Republic of Tatarstan from a non-bacterial economy. Obtained by bacteriological and biological research of pathological material by the method of selecting vital sections with their subsequent microscopy, staging a bioassay on rabbits and isolating a pure culture of the causative agent of necrobacteriosis on a hepatic nutrient medium (Kitta-Tarozzi medium) under anaerobic conditions. In smears of fingerprints from pathological material, it has the form of long, sometimes interwoven, unevenly colored gram-negative filaments, reaching a length of 10 to 100 microns, or sticks. They are motionless and do not form a spore. Colonies on an agarose medium are dewy, silvery, convex, transparent, round, with a diameter of 2-4 mm; on blood agar, a hemolysis zone of 8-10 mm agglutinates red blood cells of chickens at a dilution of 1: 128. The strain has sufficient immunogenicity, so in an indirect immunofluorescence reaction with homologous serum, it reveals an antigen at a dilution of 1: 5180. The strain was identified by the Bergi microorganism determinant as a species of Fusobacterium necrophorum and a subspecies subspecies necrophorum according to the following genes: sheath protein (RNA polymerase beta subunit (rpoB) in 906 bp, hemagglutinin (outer membrane protein gene) in 540 np, DNA gyrase (gyrase B subunit) at 427 np, transposase (repeated DNA sequence) at 557 np using PCR and subsequent sequencing of the corresponding fragments. Bacterial strain NII KKM SSC VB "Vector" B-1076 Fusobacterium necrophorum subspecies necrophorum deposited at the Research Institute “Collection of microorganism cultures” FSUE SSC WB “Vector” under the number Research Institute KKM SSC WB “Vector” B-1076 dated September 8, 2005.

The isolated bacterial strain of necrobacteriosis causes a chronic infectious disease characterized by purulent-necrotic lesions of the skin, mucous membranes, limbs and parenchymal organs.

Cultural and morphological features of the strain: gram-negative filaments and sticks of different lengths, non-spore-forming, motionless, arranged in the form of plexuses, colonies on an agarose medium are dewy, silvery, convex, transparent, round, with a diameter of 2-4 mm; on blood agar, a hemolysis zone of 8-10 mm agglutinates chicken red blood cells in a dilution of up to 1: 128. Produces indole and hydrogen sulfide. Ferments with the formation of butyric acid and a small amount of gas glucose, sucrose, maltose, beckons, dulcin, inulin. Weakly ferment lactose. It forms a thermolabile exotoxin, causing the death of white mice on 1-2 days after their intraperitoneal infection.

The activity (productivity) of the strain (indicating the cultivation conditions), other production indicators: in the Kitta-Tarozzi environment it grows at + 37 ° C for 24 hours in the form of profuse turbidity, at the beginning in the lower layers, and then the whole medium. In subsequent cultivation, a loose precipitate precipitates and the medium becomes transparent.

A method for determining the activity of a strain indicating the method. With subcutaneous infection with a daily broth culture, white mice die on the 4th-5th day, rabbits on the 8th-10th day with a pronounced purulent-necrotic tissue lesion at the injection site and extensive lesions of the parenchymal organs (lungs, heart, liver). In RNIF with hemologic serum, the antigen titer is 1: 5180, with heterologous 1: 320-1: 640.

The method, conditions and composition of media for long-term storage of the strain: lyophilization in its native form in Kitta-Tarozzi medium with the addition of 10% normal cattle serum, as well as by freezing in a nutrient medium with glycerol.

Method, conditions and composition of propagation media: Kitta-Tarozzi medium supplemented with 10% normal cattle serum. The conditions and composition of the medium for fermentation: under the conditions of a thermostat in large bottles - stationary and in reactors in a medium prepared on the basis of the Hottinger pass under liquid paraffin with the addition of 10% normal cattle serum, at a temperature of 37 ° C and pH 7.2-7 , 6.

Genetic features of the strain: prototroph, uses normal serum as a growth factor, is resistant to streptomycin, monomycin, neomycin.

The invention is illustrated by the following examples.

Example 1. The selection of the strain. The strain deposited in the Research Institute of KKM SSC VB "Vector" under the number B-1076 Fusobacterium necrophorum subspecies necrophorum was isolated from a sick first-calf from a farm unsuccessful for necrobacteriosis. With a purulent-necrotic process in the finger area, dead tissue is removed. At the border of living and necrotic tissue, hooves are scraped. Dabs are made from scrapings and stained according to Romanovsky-Giemsa and Gram. Gram-negative filaments of different lengths or sticks were visible in smears from pathological material. Such scrapings are subjected to bacteriological and biological research.

During bacteriological examination, a 10% suspension is made from pathological material on sterile physiological saline or meat and peptone broth and plated on Kitta-Tarozzi medium with the addition of up to 10% of normal cattle serum. Before sowing, the Kitta-Tarozzi medium is regenerated by heating in a boiling water bath for 20-30 minutes, after which it is quickly cooled to 45-50 ° C. Grow the culture in a thermostat at a temperature of + 37 ° C for 5 days with daily viewing. After 14-24 hours in the Kitta-Tarozzi medium, Fusobacterium necrophorum forms a filamentary haze, initially in the lower layers of the medium, and later in the upper layers, gas formation is very weak. On day 5, broth enlightenment occurs, with sediment precipitating at the bottom of the tube. Microscopic examination of smears from the culture reveals granularly colored long interwoven filaments, which in some cases have bulb-like extensions.

Bacteriological research is carried out to obtain a pure culture. For this purpose, along with sowing on a nutrient medium, the rabbit is infected. To do this, infect the original 10% suspension of pathological material or daily broth culture under the skin of the middle third of the outer surface of the ear in a dose of 0.5-1.0 ml. Observation of infected animals is carried out for 10 days.

In the presence of Fusobacterium necrophorum in the pathological material or the studied culture, dry necrosis develops at the injection site at the injection site after 4-7 days. From the focus of necrosis, seeding on the Kitta-Tarozzi medium and smears are done, which are stained according to Gram. If granular stained filaments are found in smears, the bioassay is considered positive, and the Fusobacterium necrophorum culture is isolated.

Thus, the use of microbiological and biological methods makes it possible to isolate the culture of Fusobacterium, the causative agent of animal necrobacteriosis, and to identify it as a species of Fusobacterium necrophorum.

Example 2. Genotyping of a strain. Polymerase chain reactions are carried out by the synthesis of DNA fragments of various genes of the strain. As primers, the first and last 18-20 nucleotides below the nucleotide sequences are used. Then, the synthesized DNA fragments of the corresponding gene fragments are sequenced: envelope protein, hemagglutinin, DNA gyrase and transposase. (Fig. 1-4) The determination of the nucleotide sequence of the fragments is carried out using two DNA strands using the generally accepted Maxam-Gilbert technique. When analyzing the nucleotide sequences of fragments of the above genes with the prototype, it was found that DNA fragments in 906 bp of the rpoB gene (RNA polymerase beta subunit), in 540 bp of the ompH gene (outer membrane protein gene) strain of the Research Institute of CMC SSC WB " The vector "B-1076 coincides with the sequence of Fusobacterium necrophorum subsp.necrophorum units ATCC25286 (AF527637).

The hemagglutinin gene is absent in Fusobacterium necrophorum subsp.funduliforme and Fusobacterium nucleatum. The nucleotide sequence of 427 N. p. of the gene fragment of the DNA gyrase enzyme (gyrase B subunit) of the research institute of KKM SSC VB "Vector" B-1076 coincided with the nucleotide sequence of the strains Fusobacterium necrophorum subsp.necrophorum NCTC10576 (AY372007), Fnl6fumorumfumorumfumorumfumorumfyrumfumorumfumorumfumorumfumorumfumorium ) The nucleotide sequence of DNA in 557 N. p. the transposon gene (repeated DNA sequence) of the research institute of CMC SSC VB "Vector" B-1076 coincides with the nucleotide sequence of the FnS1 strain Fusobacterium necrophorum subsp.necrophorum (CAA48747, NCIBI), which confirms its relation to the Fusobacterium family. Based on the results of genotyping according to the genes of the envelope protein, DNA gyrase, the strain NII KKM SSC VB “Vector” B-1076 is assigned to the species Fusobacterium necrophorum and according to the hemagglutinin and transposon genes to the subspecies necrophorum.

To further confirm the belonging of the strain deposited at the Research Institute of KKM SSC VB "Vector" under the number B-1076 to Fusobacterium necrophorum subsp.necrophorum, polymorphism of restriction fragment lengths (RFLP) of the synthesized transposon gene is studied. To this end, synthesis is carried out using nested primers of an internal fragment of 289 bp, using nested primers according to the protocol described in "Method for the detection of Fusobacterium necrophorum in nested PCR" No. 2203951 from 05.10.03. The endonuclease MspI (C'CGG) amplicon is hydrolyzed and analyzed by electrophoresis in 2% agarose. Get a genetic profile consisting of two fragments with a length of 170 and 119 N. p. The marker is pUC18 hydrolyzed AluI DNA. The resulting genetic profile of the internal fragment of the transposon confirms that the strain of the Research Institute of KKM SSC VB "Vector" B-1076 belongs to Fusobacterium necrophorum subsp.necrophorum.

Thus, the genotyping performed allows us to identify the culture isolated using microbiological and biological methods as a strain of Fusobacterium necrophorum subspecies necrophorum SRI KKM SSC VB "Vector" V-1076.

Example 3. According to the results of genotyping of the strain Fusobacterium necrophorum subspecies necrophorum deposited at the Research Institute of KKM SSC VB "Vector" under the number B-1076, a "Method for the detection of Fusobacterium necrophorum in nested PCR" No. 2203951 dated 05.10.03 was developed, based on which " Test system for the detection of Fusobacterium necrophorum subsp.necrophorum by polymerase chain reaction (PCR) using nested primers ", which passed state commission tests.

Example 4. For the manufacture of a diagnostic test system that identifies both pathogenic subspecies of Fusobacterium necrophorum, primers are selected in the sequences common to these subspecies — this is the DNA sequence of the GroV gene encoding one of the cell membrane proteins or the DNA gyrase sequence. As primers use 18-20 nucleotides of the DNA sequence. One of them is direct in the region of 5 ', and the other inverse is in the region of 3' of the end, so that between the primers there is a fragment of 300-500 nucleotide pairs.

Isolation of Fusobacterium necrophorum DNA from biological samples. A 20% suspension of 0.9% NaCl is prepared from a scraping fragment from the affected area of the hooves. Then, to the 50 μl suspension, add 500 μl of heated 10% CTAB, mix and incubate at 80 ° C for 2 hours. Then, phenol / chloroform / isoamyl alcohol (25: 24: 1) was added in half the volume of the mixture and centrifuged for 10 minutes at 13000 rpm. The aqueous phase is transferred to another tube and add 1/10 volume of 3 M sodium acetate pH 4.8 and 2 volumes of ethanol and incubated for 40 minutes or night at -20 ° C. DNA precipitate is precipitated by high speed centrifugation. The DNA pellet is dissolved in 20-30 μl of sterile double-distilled water or 10 mM TE buffer pH 8.0.

The polymerase chain reaction was carried out for 27 cycles in the following mode: preliminary DNA heating at 95 ° C for 3.0 min - 1 cycle; then 25 cycles of three segments each: DNA denaturation at 94 ° C for 0.3 min, hybridization at 56 ° C for 0.4 min, elongation at 72 ° C for 0.5 min and 1 cycle - dosynthesis at 72 ° C for 1.2 minutes The positive result of the analysis is judged by comparing to the size in the positive DNA control, which must match.

Example 5. If only the more pathogenic subspecies Fusobacterium necrophorum subsp.necrophorum is detected, the primers are selected based on the DNA sequence of the hemagglutinin gene. The remaining operations are similar to those described in example 4.

Example 6. The manufacture of a vaccine preparation for the prevention of animal diseases with necrobacteriosis includes the following steps:

1. Preparation of matrix seed. 1-2 ml of the culture of this strain is introduced into a 200 ml bottle containing 80-100 ml of Kitta-Tarozzi medium, and 0.1 ml each in a test tube with the same medium. Crops are cultivated for 18-20 hours at + 37 ° C. The grown culture is checked for cleanliness by making a smear and staining according to Gram.

2. A pure culture of the vials is seeded in a 10-liter bottle (or reactor) with Kitta-Tarozzi medium, filled to half the volume. In parallel, inoculations were again performed in vitro with Kitta-Tarozzi medium for purity control. The culture in the bottle is grown with gentle stirring with the addition of 40% glucose to a final concentration of 0.2% at + 37 ° C and a pH of 7.6.

3. After growing, determine the concentration of microbial bodies in the culture according to the optical turbidity standard and the purity of the culture by seeding in tubes on Kitta-Tarozzi medium to control purity. The grown bacterial mass in the bottle is diluted with sterile saline to a concentration of 10 billion microbial bodies in 1 ml.

4. Formalin diluted with physiological saline 1: 1 is added to the bacterial mass with constant stirring so that its concentration in the culture is 0.3% and thimersal in the amount of 0.1 g per liter of culture. Inactivation of the culture is carried out for 15-16 days at + 37 ° C. Twice a day, the culture is mixed for 5 minutes. After 12 days, a sample of the bacterial mass is taken to check for cleanliness (a smear is prepared) and inactivation for completeness by seeding on a Kitta-Tarozzi culture medium. Analysis is carried out after 8 days.

5. To the inactivated culture add a sterile 6% solution of aluminum hydroxide to 30% by volume and 0.05 g of thimersal per 1 liter of vaccine and mix every 1.5-2.0 hours for the first 10-12 hours. The pH of the vaccine is then checked and adjusted to 7.2-7.4 with a 10% sodium hydroxide solution. A sample is taken to check for sterility and safety.

6. The vaccine tested for sterility and safety is packaged in bottles with constant stirring. Close with rubber stoppers and roll up with metal caps.

7. The vaccine is used to vaccinate animals for prophylactic purposes in places unsuccessful for necrobacillosis of cattle. The vaccine is injected into the muscles in animals at a dose of 2 ml. Immunity in vaccinated animals occurs 14 days after vaccination and lasts up to 6 months.

Thus, from examples 1-5 to verify the proposed strain of Fusobacterium necrophorum subspecies necrophorum deposited at the Research Institute of KKM SSC VB "Vector" under the number B-1076 shows that the invention can be used in agriculture for the manufacture of diagnostic and prophylactic drugs against necrobacteriosis animals.

Claims (1)

The bacterial strain Fusobacterium necrophorum subspecies necrophorum, deposited in the Research Institute of KKM SSC WB "Vector" under the number B-1076 for the manufacture of diagnostic and prophylactic drugs against necrobacteriosis of animals.

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