RU2544951C1 - ATTENUATED STRAIN Bacillus anthracis FOR DEVELOPING MEANS OF SPECIFIC PREVENTION OF ANTHRAX - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: strain of B. anthracis 363/11 is deposited in the collection of strains of microorganisms of State Scientific Institution of Russian National Research Institute of Veterinary Virology and Microbiology of Russian Agricultural Academy No. 363. The strain is made for developing the means of specific prevention of anthrax.

EFFECT: invention enables to expand the range of protective action, to increase immunogenicity, to reduce immunising dose and reactogenicity of the vaccine strain against anthrax.

5 tbl, 5 ex

Description

The invention relates to the field of veterinary microbiology, relates to a capsule-free naturally attenuated strain of Bacillus anthracis 363/11 and can be used in research and biotechnological centers to create specific prophylaxis of anthrax.

Due to the widespread prevalence of anthrax in the Russian Federation in the past and the ability of anthrax microbial spores to remain in the soil foci for dozens or even hundreds of years, well-being for this disease is mainly maintained by conducting universal vaccination of agricultural animals. Despite this, in Russia annually from 2 to 16 sporadic cases of anthrax are recorded among susceptible animals, most of which underwent preventive immunization. Among the main causes of outbreaks of anthrax can be attributed to ineffective vaccinations, violation of the storage conditions of biological products and the circulation in nature of isolates that are not immunologically relevant to the vaccine strains used [Yu.O. Selyaninov, I.Yu. Egorova, 2012].

Currently, in the Russian Federation, a live vaccine from the capsule-free avirulent vaccine strain 55-VNIIIVViM and in public health from the capsule-free vaccine strain STI-1 are used to ensure biosecurity against anthrax infection in animal husbandry. Abroad, vaccines from B. anthracis Sterne strains (34 F ₂ ), Weybridge (Europe, USA, etc.), Ihtiman (Bulgaria), K79Z (Ukraine), 1190-R (Romania) are used to prevent this disease in animals and humans. and others [G.G. Onishchenko et al., 1999]. All of the vaccine strains listed do not contain the pXO2 plasmid, which is responsible for capsule production, the main pathogenicity factor of the anthrax microbe.

Obtaining strains of B. anthracis, promising to create live vaccines based on them, was carried out in two main ways: by artificial elimination of the plasmid pXO2 in weakened capsule-forming strains, followed by selection of capsule-free variants and selection of capsule-free strains of B. anthracis from naturally weakened field isolates.

The vaccine strains 1190-R (1934), Sterne (1937), Weybridge (1939), Takahaschi (1939), Makteswar (1939), STI (1940), NIIEG K-II (1940) were obtained from virulent capsule-forming isolates of the anthrax microbe ), Shuya-15 (1949), the second is the Ihtiman strain and 55-VNIIVViM [E.N. Shlyakhov, 1960; RS Slavtchev, 1967; G.G. Onishchenko et al., 1999]. However, a comparative study of the specific activity of the most well-known vaccine strains (STI, GNOC, Ihtiman, 34-F ₂ , 55-VNIIVViM) revealed a significant and significant difference in their immunogenicity and protective spectra with respect to some field isolates, up to complete exposure to infection [I.A. Bakulov, V.A. Gavrilov, 1991]. To overcome these shortcomings, the authors propose using chemical vaccines based on protective antigen, combined vaccines based on spores of one strain and protective antigen of another strain, divalent and trivalent vaccines based on different strains, and also to search for new strains that have the most complete antigenic composition.

A close analogue of the present invention is the capsule-free avirulent strain 55-VNIIVViM, obtained by selection in VNIIVViM from isolate C-63, isolated from the Kharkov Veterinary Institute from a pig corpse in 1963, and used to make a vaccine against anthrax in animals. Strain 55-VNIIVViM in grafted doses of 12.5 × 10 ⁷ and 25.0 × 10 ⁷ spores induces the generation of intense anti-anthrax immunity in the body of vaccinated animals (small and cattle, respectively) and protects from 40 to 100% of the livestock from infection by field virulent isolates [I.A. Bakulov, V.A. Gavrilov, 1991].

The disadvantages of the strain are its low antigenicity, an insufficiently wide range of protective actions in relation to field isolates circulating in Russia and residual reactogenicity. When receiving anthrax hyperimmune sera using 4 different immunization schemes, antibody titers to antigens of strain 55-VNIIIVViM in all cases were 2-8 times lower than to antigens of strain Sterne (34F ₂ ). A study of the genomic polymorphism of museum cultures of B. anthracis by GNU VNIIVViM employees showed that some of them have unique genotypes that differ significantly from the molecular types of the majority of the studied isolates of the anthrax pathogen, including strain 55-VNIIIVViM. So, the isolate isolated in the Udmurt SSR in 1989, which differs in genotype from strain 55-VNIIVViM, caused cattle disease one month after it was immunized with a vaccine from strain 55-VNIIVViM. The presence of reactivity of the vaccine from strain 55-VNIIIVViM is evidenced by reports of veterinarians about post-vaccination complications in goats - the formation of serous edema at the injection site.

The aim of the invention is to obtain a new avirulent strain of Bacillus anthracis, which has a wider spectrum of protective action, greater immunogenicity and less, compared with the analogue strain, reactogenicity and immunizing dose.

The goal is achieved by isolating a new naturally attenuated capsuleless strain of anthrax microbe. The inventive strain 363/11 isolated by employees of the GNU VNIIVViM in 2011 from the dead gilt.

Strain 363/11 was deposited in the Collection of microorganism strains of GNU VNIIVViM of the Russian Agricultural Academy under inv. No. 363.

Strain Bacillus anthracis 363/11 is characterized by the following cultural-morphological, immunobiological and biochemical characteristics.

Cultural properties. Optional aerob. Optimum growth temperature on solid and liquid nutrient media (36 ± 1) ° C, optimum pH (7.4 ± 0.2). It has cultural and morphological properties typical of B. anthracis species: upon cultivation in liquid nutrient media, the broth remains transparent, a sediment in the form of a cotton ball is formed at the bottom; on solid nutrient media forms matte, rough colonies of R-, less often RO-form, with a diameter of 3-4 mm. On media containing horse serum and in the presence of CO ₂ , as well as in the body of susceptible laboratory animals, it does not form a capsular polypeptide.

Morphological properties. It exists in two morphological forms: vegetative and spore. In smears from culture media, gram-positive sticks are arranged in the form of long chains, which at their junctions look chopped off or slightly concave. Spores painted according to Ziehl-Nielsen have an oval shape, are located mainly centrally. Cells of flagella do not have, motionless.

In vivo (in the organism of white mice) forms chains consisting of 4, 8, 16, 21 and more segments.

Biochemical properties. Causes ram hemolysis of sheep erythrocytes. Ferments with the formation of acid without gas cellobiose, sucrose, trehalose. Does not decompose arginine. Oxidizes glucose to acetoin. Restores nitrates to nitrites. It produces catalase, does not form alkaline phosphatase.

Antigenic properties. The somatic antigen of strain 363/11, obtained by thermoextraction, interacts specifically with anthrax precipitating serum, and the daily broth culture supernatan interacts with globulin antisybrasyl from horse blood serum.

When a spore suspension is administered subcutaneously to sheep, the strain induces the production of agglutinating antibodies.

Weakly sensitive to the effects of the anthrax phage Fah-VNIIVViM and highly sensitive to the phage Rd / Ph / 6.

Plasmid composition. It contains pag gene sequences in the genome, the genes of the capsular operon are absent (pXO1 ⁺ pXO2 ^- ).

Residual virulence. LD ₅₀ for outbred white mice with a live weight of 18-20 g is 3.162 × 10 ⁵ spores, for guinea pigs with a live weight of 200-300 g - over 10 ⁸ spores.

Reversibility. It does not restore the ability to capsule formation for 7 consecutive passages in guinea pigs (at passage 8 it ceases to multiply in the body of guinea pigs) and 10 passages in outbred white mice.

Reactogenicity. Weakly reactive for guinea pigs: with subcutaneous administration of 10 ⁷ spores, serous edema forms at the injection site, rarely with subsequent ulcerations. Not reactogenic for sheep with the introduction of 10 ⁷ spores subcutaneously in the area of the hairless area of the armpit.

Immunogenic properties. In a graft dose of 10 ⁶ -10 ⁷ spores cause the formation of vaccinated animals (guinea pigs, sheep) of intense immunity against capsule-forming reference infecting strains (II Tsenkovsky vaccine, 17JB) and field isolates.

To confirm the advantages of the present invention, examples of its specific implementation are given.

Example 1. Description of the main biological properties of the vaccine strain 55-VNIIVViM and capsule-free naturally attenuated strain 363/11.

The biological characteristics of strains 55-VNIIVViM and 363/11 are presented in table 1.

Table 1 Characterization of the biological properties of strains 55-VNIIVViM and 363/11 No. p / p Signs Strain characteristics 55-VNIIVViM 363/11 one. In vitro morphology sticks, spores sticks, spores 2. In vivo morphology sticks connected in chains of 2, 4, 6 and sometimes 8 members sticks connected in chains of 4, 8, 16, 21 and more segments 3. Mobility absent absent four. Gram stain gram positive gram positive 5. BCH Growth “Cotton ball” in a transparent broth, formation of a parietal ring on the 5th day "Cotton ball" in a clear broth 6. Solid Growth R-shapes with typical “curls” R and RO-forms with typical “curls” 7. Type of hemolysis, exotoxin production level β-hemolysis, medium high hemolysis 8. Sensitivity to phage Fah-VNIIVViM weakly sensitive weakly sensitive 9. Phage Sensitivity Rd / Ph / 6 sensitive (#) sensitive (#) 10. Capsule availability absent absent eleven. In vitro and in vivo toxin production produces produces 12. The presence of the pag gene is available is available 13. The presence of the cap gene absent absent

The data presented in table 1 indicate the presence of properties typical for vaccine strains in both strains (persistent loss of capsule formation). The differences between the strains are the ability to express in vitro different types of hemolysins at different levels.

Example 2

The residual virulence of strains 55-VNIIVViM and 363/11 was determined on the model of outbred white mice with a live weight of 18-20 g and guinea pigs with a live weight of 200-300 g by subcutaneous inoculation of the latter in a volume of 0.25 cm ^{3 of} spore suspensions of serial dilutions of pathogens prepared with in five steps. Observation of animals was carried out for 10 days. At the end of the observation period, the ratio of the number of animals that died from the administration of a given dose to the total number of animals to which this dose was administered was determined. The value of LD _{50 was} calculated according to the Kerber formula in the modification of I.P. Ashmarina and A.A. Vorobyov (Table 2).

table 2 Comparative assessment of residual virulence of strains 55-VNIIVViM and 363/11 Animal species LD ₅₀ values for strains, spores 55-VNIIVViM 363/11 Outbred White Mice 115.0 × 10 ⁵ 3.162 × 10 ⁵ Guinea pigs over 10 ⁸ over 10 ⁸

The data in table 2 indicate that both strains are characterized by similar indicators LD ₅₀ for guinea pigs and belong to the group of avirulent strains. In terms of LD ₅₀ for outbred white mice, strains 363/11 and 55-VNIIIVViM have differences, but according to the classification of E.N. Shlyakhova and E.V. Cargo (1978) both strains also belong to the group of avirulent cultures.

Example 3

In experiments on guinea pigs, the parameters of immunizing doses of strains 55-VNIIVViM and 363/11 were determined, which protect against the death of 50% of vaccinated animals (ImD ₅₀ ) after infection with a reference-infecting culture of anthrax microbe strain No. 71/12 at a dose of 200 LD ₅₀ . The results of a comparative determination in guinea pigs of ImD ₅₀ strains of B. anthracis 55-VNIIVViM and 363/11 are given in table 3.

Table 3 Indicators ImD ₅₀ for guinea pigs of strains of B. anthracis 55-VNIIVViM and 363/11 Indicator Strain 55-VNIIVViM Strain 363/11 ID ₅₀ for guinea pigs in relation to the reference infecting strain No. 71/12 (2.9-3.4) × 10 ⁵ spores 2,951 × 10 ⁴ spores

The data in table 3 indicate that the index of ImD ₅₀ for strain 363/11 is 10 times lower than for strain 55-VNIIIVViM, i.e. to induce protective immunity in guinea pigs, which protects 50% of vaccinated animals from death after the introduction of spores of strain No. 71/12 at a dose of 200 LD ₅₀ , a 10-fold lower vaccination dose is required.

Example 4

In experiments on guinea pigs, it was shown that the protective spectrum of strain 363/11 is wider than strain 55-VNIIVViM. Survival percentage of guinea pigs vaccinated with 363/11 strain infected with reference infecting cultures of anthrax microbe (No. 71/12, 17JB, 76) and field isolates isolated in different regions of Russia and at different times (No. 81, 304, 364) was higher than for guinea pigs vaccinated with strain 55-VNIIIVViM (Table 4).

Table 4 Immunogenic properties of strains of B. anthracis 55-VNIIVViM and 363/11 in tests on guinea pigs Strain B. anthracis Immunizing dose, spore The percentage of protection of vaccinated animals from infection with various anthrax strains (infectious dose of 200 LD ₅₀ ) 71/12 76 81 304 364 ** 17JB 363/11 10 ⁷ 80 one hundred* one hundred* fifty 40 one hundred 55-VNIIVViM 1.2 × 10 ⁷ 80 one hundred*** 40 *** 25 10 one hundred Note: * - the vaccine dose of the strain was 10 ⁶ spores; ** - the infecting dose of the strain was 1200 LD ₅₀ ; *** - data of Bakulov I.A. and Gavrilova V.A. (1991)

From the data presented in table 4 it is seen that vaccination of animals with strain 363/11 protects 100, 50 and 40 percent of guinea pigs from infection with cultures of virulent field isolates of B. anthracis strains Nos. 81, 304, 364, respectively, while the percentage of protection vaccinated animals with strain 55-VNIIVViM for these field isolates is 40, 25 and 10%, respectively. The immunogenic activity of strains 363/11 and 55-VNIIVViM in relation to reference infecting cultures of strains No. 71/12, 17JB, 76 had no differences.

Example 5

The immunogenic activity of the strains was determined in experiments on sheep aged 1-1.5 years from a herd that had not been vaccinated against anthrax. Sheep was injected under the skin with 10-12 million spores of the tested strains in a volume of 1 ml.

After 21 days, the vaccinated sheep were infected by intracutaneous administration of spore cultures of highly virulent anthrax strains in doses of at least 10 unconditionally deadly doses for the sheep. Observation of infected animals was carried out for 10 days. Sheep, which developed intense immunity against the corresponding infecting strain, remained clinically healthy. Unprotected animals died within 3-5 days after infection with signs of acute anthrax. The specific nature of the death of sheep was confirmed by microscopy of blood smears and bacteriological analysis.

Table 4 Immunogenic properties of strains of B. anthracis 55-VNIIVViM and 363/11 in sheep Strain B. anthracis Immunizing dose, spore The strains used for infection (200 LD ₅₀ ) Number 76 №304 Number 81 363/11 10 ⁷ survived survived survived 55-VNIIVViM 1.2 × 10 ⁷ survived fell fell

The data in the tables indicate that the spectrum of the protective effect of strain 363/11 is wider than that of strain 55-VNIIVViM. Those. immunization of animals with strain 363/11, unlike strain 55-VNIIVViM, protects them from infection with field isolates of B. anthracis immunologically not corresponding to strain 55-VNIIVViM (Nos. 304 and 81).

Information sources

1. Ashmarin, I.P. Statistical methods in microbiological research / I.P. Ashmarin, A.A. Vorobiev. - L .: Medgiz, 1962. - P.85.

2. Bakulov, I.A. Immunoprophylaxis of anthrax animals / I.A. Bakulov, V.A. Gavrilov // News, S.-kh. Sciences. - 1991. - No. 8. - S.128-131.

3. Selyaninov, Yu.O. About some reasons for the lack of effectiveness of measures for the specific prevention of anthrax in the Russian Federation / Yu.O. Selyaninov, I.Yu. Egorova // Actual problems of diseases common to humans and animals: Mater. Vseros. scientific-practical conf. from the international participation - Stavropol. - 2012. - P.69-70.

4. Anthrax: current aspects of microbiology, epidemiology, clinic, diagnosis, treatment and prevention / G.G. Onishchenko [et al.] - M .: VUNMTS Ministry of Health of the Russian Federation, 1999. - 448 p.

5. Shlyakhov, E.N. Epidemiology, diagnosis and prevention of anthrax / E.N. Shlyakhov. - Chisinau: Cartya Moldovenienasca, 1960 .-- 116 p.

6. Shlyakhov, E.N. Titration of LD ₅₀ in mice as a method for determining the virulence of anthrax bacilli / E.N. Shlyakhov, E.V. Cargo // Achievements and prospects of the fight against anthrax in the USSR: Mater. X plenary session of the interagency anthrax commission. - M., 1978. - P.97-99.

7. Slavtchev, R.S. Etude comparative de la propriete oedematogene Lune nouvelle souche acapsulogene de Bacillus anthracis (souche Ihtiman) / R.S. Slavtchev // Arch. Inst. Pasteur, Tunis. - 1967. - V.44, No. 2-3. - P.229-233.

Claims (1)

The attenuated B.anthracis strain 363/11 for the development of specific anthrax prophylaxis agents was deposited in the Collection of Microorganisms of the All-Russian Scientific Research Institute of Veterinary Virology and Microbiology of the Russian Agricultural Academy under No. 363.