UA39746U - Vaccinating strain of bacteria bacillus anthracis sb againts animal anthrax - Google Patents

Abstract

A vaccinating strain of bacteria Bacillus anthracis SB relates to biotechnology, in particular to veterinary microbiology, in particular to t a vaccinating strain for preparation of bacterial preparation – animal anthrax vaccine.

Description

Host-related cues that affect the transcription of toxin and capsule genes include temperature and CO concentration.'

Characterization of the SB strain by the presence of plasmid and chromosomal markers by the polymerase chain reaction method: pXO17 (RA", GE, ERU; pxo2 (Sar). The SB strain does not carry the pxo2 plasmid and, therefore, is not capable of synthesizing a capsule.

Oligonucleotide primers with the sequence for the complete RA gene were used to detect the рхХОЙ plasmid (genes of the protein factors of the anthrax toxin): direct - 5-

STATATOAADAAAASOSAAAAASTaTTTAATASOS-3" and reverse - 5- SATSOSTASAAAASAATSTSAAASO-3.

To detect capsule genes, oligonucleotide primers with the following sequence were used: direct - 5'-

SstTaAasSSATTAATSSATATO-3: and the reverse - -TSSSASTTASOITAATSTOada-3.

To check (verify) the results obtained with the RA gene, a gene fragment was additionally amplified

RA size Obp.n. with primers: forward - 5-ТССТАСАСТААСОАА(ТОС(-3 and reverse - 5- вАаатТАСААдОасСАТАТАСОСТ-В' followed by a restriction analysis with the enzyme Mbro 1/ The size and number (three) of the obtained fragments in SB, 55 and 34 E coincided.

The proposed vaccine strain of bacteria Vasiya5 apiphagesia SB, which is offered, is characterized by the following physiological signs:

Morphology of cells: under microscopy of Gram-stained preparations made from cultures on MPA - straight rods of purple color (5-8x0.8-1μm), single or in chains of different lengths; without capsules; spores are oval, colorless, formed in the center of cells.

Mobility: sticks are immobile.

Morphology of colonies on solid nutrient media: A-form colonies, grayish-white, rounded, convex, with a wavy edge and a granular mass inside.

Cultural features on liquid nutrient media: by visual assessment of the daily culture of the strain v

MPB (pH 7.2-7.4): a whitish loose sediment forms at the bottom of the test tube, the broth above the sediment is transparent, the film and wall ring do not form. On the medium with bovine blood serum: growth throughout the column in the form of clouds; more significant sediment is formed than on MPB.

Enzymatic activity: ferments sucrose, glucose with acid release without gas release; does not ferment mannitol, lactose, maltose; decolorizes methylene blue for 30 minutes or more. When sowing on blood agar after 24 hours, there is no hemolysis, later - the presence of zones of hemolysis.

Sensitivity to antibiotics: most sensitive to ampicillin, erythromycin, tetracycline, chloramphenicol, gentamicin, cefaclor, amikacin, doxycycline, impenem, less sensitive to neomycin, streptomycin, nystatin, olendomycin. Insensitive to amphotericin B, introconazole, ketoconazole, polymyxin, cefepin, ceftibuten.

Biological properties: when administered intraperitoneally to white mice weighing 18-20 g, I O5o contains 12.4-12.6x106 live microbial cells. Subcutaneous administration of 1 cm3 to rabbits weighing 2-2.5 kg: 170 mln. of living microbial cells does not cause their death, and at the injection site there is an increase in temperature during the first three days and swellings of 1x1.5 cm, which later disappear.

Capsule formation: in the organs of affected animals and on GKI media with 4095 serum content of GKI is absent.

There are no capsules around the vegetative cells. When cultivating the Vasiy5 apiygasiz SB strain by the surface method in 4-liter bottles, the accumulation reaches 2.0-2.4 billion. dispute on a bottle.

Storage: the culture of the proposed strain is stored in a lyophilized state or in the form of a spore suspension on glycerol.

The proposed strain has special properties: low virulence, which is characterized by an IU5o value above 5.0x102 live microbial cells, namely: 12.4-12.6x109 live microbial cells with significant immunogenicity. At the same time, the productivity of the strain during surface cultivation reaches 2.4 billion. spores per bottle, while the mass fraction of spores is 99-10095. As research has shown, along with a low level of virulence and, accordingly, a low level of reactogenicity, the new strain has a pronounced resistance to antibiotics - amphotericin B, inraconazole, ketoconazole, polymyxin, cefepin, ceftibuten, that is, it is not characterized by sensitivity to these antibiotics. Such properties of the strain make it possible to improve the quality of the anthrax vaccine, to expand the scope of vaccine use, to use vaccination of animals in necessary cases in combination with their treatment with antibiotics, and at the same time, it is possible to combine the insensitivity of the strain culture to certain antibiotics.

Examples of strain reproduction.

Example 1. The spore culture of the Vasiyshe apipagsiae SB strain is obtained by cultivating the bacteria Vasishev apipagsiae SB on a sterile nutrient medium - meat-peptone agar in a 4-liter bottle. The bottle is filled with nutrient medium in the amount of 150 cm per bottle. Such an environment is distributed over the inner surface of the cylinder in a thin layer with simultaneous cooling of the environment. Then the nutrient medium is kept for 24 hours at a temperature of 3724170 for the purpose of checking for sterility, after which it is inoculated with a bacterial suspension of Vasiyshe apigasi5 SB and kept for two days at a temperature of 3731"C, and then for five days at a temperature of 325217C. The spores that have formed are washed off from the surface of the agar, 100 ml of sterile distilled water and concentrate to the required level. The biomass accumulation of the new strain is 2.0-2.4 billion spores per bottle, while the mass fraction of spores is 99-10095. To stabilize the number of microbial cells, the spore suspension is mixed with 30 -3395 glycerol The finished vaccine is stored at a temperature of 2-1576.

The obtained culture of the Vasiyn5 apingasis SB strain is tested for typicality, uniformity, capsule formation, residual virulence, harmlessness, immunogenicity, sensitivity to antibiotics. This culture is characterized by the following parameters: typicality - cultural and morphological properties typical for the strain; homogeneity - according to the type of colonies, the number of dissociated A-form colonies does not exceed 5,905, the absence of capsules in smears - prints from the organs of animals that died from the introduction of increased doses of the VasshShyvye apipgasiye SB strain.

The virulence of the obtained culture, which was determined on white mice weighing 18-20 g, is characterized by the value of I O5o of 12.5x106 live microbial cells. Sensitivity to penicillin is determined by the method of paper discs with antibiotics. This strain is most sensitive to erythromycin, tetracycline, chloramphenicol, less sensitive to neomycin, streptomycin, insensitive to amphotericin, inraconazole, ketocopazole, cefepin, ceftibuten,

polymyxin The immunogenicity of the strain is determined on guinea pigs weighing 380-400 g, inoculated with the Vasiyv5 apingassie SB strain, followed by control infection with the second Tsenkovsky M-71 vaccine in a lethal dose.

The introduction of a culture of the strain VasShivz apigasie SB in the indicated doses provides protection of 90-10095 animals with 10095 death of control non-vaccinated animals.

Example 2. The spore culture of the Vasiisha apigasiz SB strain is obtained by cultivating the bacteria VasshShiv apingasiv SB as in example 1, but on a sterile yeast-peptone nutrient medium, according to the following recipe: purified yeast extract - 2g, disubstituted potassium phosphate - 0.5g, calcium chloride - 0.4g, zinc sulfate - 0.01g, copper sulfate - 0.01g, iron sulfate - 0.01g, ammonium sulfate - 2g, antifoam 0.2cm3, or polypropylene glycol - 0.1cm3, distilled water 1000cm3. When grown for 24 hours and constant aeration - C, bmmol Og/l/h., pH of the medium - 7.4-7.6.

The obtained culture of the strain is characterized by the following parameters: typicality - cultural and morphological properties typical for the strain; homogeneity - according to the type of colonies, the number of dissociated colonies of B-forms does not exceed 4,892, the absence of capsules in smears - impressions from the organs of animals that died from the introduction of increased doses of the Vasiishv5 apingasis SB strain. The virulence of the obtained culture, which was determined on white mice weighing 18-20 g, is characterized by the I Ovo value of 12.6x106 live microbial cells. The accumulation of biomass of the new strain is 2.0-2.4 billion. spores per bottle, while the mass fraction of spores is 99-10095. The strain culture obtained is most sensitive to erythromycin, tetracycline, chloramphenicol, less sensitive to neomycin, streptomycin, insensitive to amphotericin B, inraconazole, ketoconazole, polymyxin, cefepin, ceftibuten, polymyxin.

The immunogenicity of the strain is determined on guinea pigs weighing 380-400 g, inoculated with a culture of the Vasiyv5 apingasis SB strain, followed by a control infection with the second Tsenkovsky M-71 vaccine in a lethal dose.

The introduction of a culture of the strain VasShivz apigasie SB in the indicated doses provides protection of 90-10095 animals with 10095 death of control non-vaccinated animals.

Example 3. The spore culture of the Vasiyshe apigasiz SB strain is obtained by cultivating the bacteria VasshShiv apingasiv SB as in example 1, but on a sterile nutrient medium - enzymatic hydrolyzate of dry protein concentrate from the by-products of meat and dairy industries (enzymatic hydrolyzate of SBK) in a ratio with water , volume.9o: enzymatic hydrolyzate of SBK - 25, water - the last.

The obtained culture of the strain is characterized by the following parameters: typicality - cultural and morphological properties typical for the strain; homogeneity - according to the type of colonies, the number of dissociated colonies of B-forms does not exceed 4,895, the absence of capsules in smears - prints from the organs of animals that died from the introduction of increased doses of the Vasiyv5 apigasiz SB strain. The virulence of the obtained culture, which was determined on white mice weighing 18-20 g, is characterized by the I Ovo value of 12.4x106 live microbial cells. The accumulation of biomass of the new strain is 2.0-2.4 billion. spores per bottle, while the mass fraction of spores is 99-10095. The culture of the strain obtained is most sensitive to neomycin, streptomycin, insensitive to amphotericin, inraconazole, ketoconazole, cefepin, ceftibuten, polymyxin.

The immunogenicity of the strain is determined on guinea pigs weighing 380-400 g, vaccinated with the VasshShiv apingas strain

SB, followed by control infection with the second Tsenkovsky M-71 vaccine in a lethal dose. The introduction of a culture of the Vasiiinv5 apigasiz SB strain in the indicated doses provides protection of 90-10095 animals with 10095 death of control non-vaccinated animals.

Comparative characteristics of the new strain, which was obtained as shown in examples 1, 2, C, and known strains are shown in tables 2, 3, on the diagram (Fig.) and explanatory table 4 to it, while the data for table 2 were obtained by the methods samples and regression analyses.

Table 2

Comparative evaluation of the virulence of the proposed and known strains

Amount Death of one dose of cells in cm3 ' million lab 176 | 4

STI nale v rt 17777760 bi shin shi i PE shi: shi li o Prototit v ab | 8 1 80 | 50 5o (prototype) and

St. proposed) " in the

Table C

Characterization of sensitivity of strains to antibiotics

Sterile zone

AmphotericinV./: | 77777771

MPntrokanazol.o//|Y 77777771

Ketoyunazol../://|Ї 77777711

Erythromycin

Polymyxin.//////СЙГ71г

Penicilln./////// Them

Tsefklin./////// HER

Ceftibuten niiyu" shishshshzhiyazhyaiyayayaiyaiyazhzhyaiyaishyapy shi nshshsh

Table 4

Spectrum of sensitivity of strains to antibiotics shi -,. y y Number of antibiotics out of 54 to which the strains are sensitive

The largest diameter of the zone of no strain growth, mm 11111111 6 16 in1111111111111111171г1111и1111171|16 115. шин жити ши ши шш

Sources of information: 1. TUU 46.15.201.97. GOST 15 991-86. The STI vaccine is live against animal anthrax. 2. Patent Me2173342, Russian Federation, IPC C12M1/12, Ab1K39/07, application 19.01.2000, publ. 10.09.2001. 3. TU-1009-3-87. OST 10-19-002-00 Strain 55-VNYVVIM anthrax vaccine. stat 2-22 l 45-A I 5 '7

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