RU2149183C1 - Strain of bacterium brucella abortus used for preparing diagnostic and prophylactic biopreparations against brucellosis in animals - Google Patents

Abstract

FIELD: veterinary science, microbiology. SUBSTANCE: new strain of Brucella abortus N KB 13/100 is able to reproduce stably in nutrient media containing growth inhibitor of foreign microflora. Bis-triphenylanhydrocarbinol oxalate is used at concentration 0.001% as growth inhibitor. Strain shows high immunogenicity, expressed agglutinable and antigenic properties that are typical for brucella in S-form. Strain does not show pathogenicity for animals and humans. Strain Brucella abortus is deposited in microorganism collection of VGNKI at N KB 13/100 DEP. Diagnostic antigen prepared from the strain B. abortus VGNKI N KB 13/100 is the biopreparation showing high specificity and high activity and prepared vaccine shows high immunogenic activity. EFFECT: strain indicated above, valuable properties of biopreparation and vaccine. 4 tbl, 3 ex

Description

 The invention relates to the field of veterinary microbiology and is a new strain of bacteria Brucella abortus used for the manufacture of diagnostic and preventive biological products against animal brucellosis.

 A set of measures to combat animal brucellosis involves timely diagnosis and specific prevention of the disease.

 It is known that for the manufacture of a live vaccine against animal brucellosis and antigens for serological diagnosis of the disease (in RA, RSK, RDSK, RBP, KR with milk), the culture of the vaccine strain Brucella abortus 19 is used (1).

 For the manufacture of live brucellosis vaccine and preparations for the diagnosis of brucellosis, you can use the strain Brucella abortus 104M. The live vaccine from this strain is superior in specific efficacy to a similar preparation from the Brucella abortus 19 strain, and the diagnostics from strain 104M are more sensitive and specific (2). However, the disadvantage of this strain, like all other strains of brucella used for the manufacture of diagnostic and prophylactic preparations against animal brucellosis, is that when it is cultivated under industrial conditions, the bacterial mass, which is the raw material for the manufacture of vaccines and diagnostics, is often contaminated with extraneous microflora, which leads to to significant losses of raw materials (10-15%), causing significant economic damage to biological enterprises.

 The aim of the present invention is to obtain a strain of brucella intended for the manufacture of biological products for the diagnosis and prevention of animal brucellosis, which can stably reproduce on nutrient media containing a growth inhibitor of extraneous microflora with high immunogenicity, pronounced agglutinable and antigenic properties characteristic of brucella in S-form, non-pathogenic to animals and humans.

 The strain Brucella abortus was obtained as a result of targeted selection for cultural, morphological, biochemical, agglutinable, antigenic and immunogenic properties from the vaccine strain Brucella abortus 104M.

 The strain is named Brucella abortus KB 13/100, is deposited in the collection of microorganisms of the All-Russian State Research Institute for Control of Standardization and Certification of Veterinary Medicines (VGNKI) and has a registration number KB 13/100 DEPT.

 The strain Brucella abortus VGNKI N KB 13/100 is characterized by the following features and properties.

 Morphological signs.

 Brucella strain - short gram-negative rods with rounded ends, located in isolation, in pairs, less often in the form of short chains, without spores and capsules. The size is 1.0-1.2 microns, a width of 0.5-0.6 microns.

 Cultural properties.

 The strain culture grows well under aerobic conditions on dense and liquid nutrient media: meat-peptone hepatic glucose-glycerin agar (MPGGA), liver-open-hearth agar (PMA), hepatic-open-hearth agar with Hottinger digest (PMAH), potato hotting agar with (KAH), meat-peptone hepatic glucose-glycerin broth (MPGGB), liver glucose-glycerin broth (PHGB), as well as on these and other media intended for cultivation of brucella with the addition of 0.001% bis-triphenylhydrocarbinol oxalate .

On MPPGA, MPAA and other dense nutrient media intended for cultivation of brucella (4-5 days incubation at (37-38 ^o C) grows in the form of round, convex, smooth, with a clearly contoured edge, transparent colonies with a diameter of 2.0- 2.5 mm When stained with a solution of crystalline violet 1/2000 (according to White-Wilson), 100% of the colonies are stained as S-form: a light yellow center and a dark purple thin rim.When cultured on solid media containing bis-triphenylhydrocarbinol oxalate green colonies.

On MPPHGB and other liquid nutrient media intended for culturing brucella for 4-5 days of incubation at 37-38 ^o C, bacterial growth is characterized by slight opalescence of the medium and a thin parietal ring of a bluish color.

 Biochemical properties.

The strain culture grows on differential nutrient media (based on MPPGA and other solid nutrient media) containing fuchsin at a concentration of 1: 50,000, bis-triphenylhydrocarbinol oxalate at a concentration of 1: 100,000 (marker), 5 μg / cm ³ streptomycin sulfate and not grows on media containing thionine at a concentration of 1:50,000, penicillin 5 U / cm ³ , erythritol 1 mg / cm ³ .

Does not produce H ₂ S.

 Lysed by TB phage.

 Agglutinable properties.

 The strain culture is agglutinated with S-anti-brucellosis serum and not agglutinated with R-anti-brucellosis serum. The strain has a type-specific agglutinability: A is agglutinated by monoreceptor serum to its titer and not agglutinated by M serum at a 1:10 dilution.

 Antigenic properties.

When guinea pigs are given a dose of 1 • 10 ⁹ microbial cells and rabbits at a dose of 7 • 10 ⁹ microbial cells, only S antibodies are produced in animals: agglutinins from 100 to 1000 IU / cm ³ and complement binding antibodies in titers from 1:20 to 1 : 160.

 The blood serum of animals at a dilution of 1:10 does not give a positive reaction with R-brucellosis antigens.

 Harmlessness.

 Strain KB 13/100 is harmless. With the introduction of 250 million microbial cells into mice and guinea pigs, 2 billion microbial cells does not cause pathological and anatomical changes characteristic of brucellosis. Brucella strain does not migrate from vaccinated animals to intact ones.

 The strain is non-contagious. When intact guinea pigs are kept together for 90 days with guinea pigs immunized with a strain of KB 13/100 at a dose of 2 billion microbial cells, followed by bacteriological examination of brucella strain KB 13/100 from the lymph nodes and internal organs (10 objects) of unvaccinated animals stand out.

 Virulence.

The residual virulence of strain KB 13/100 is the same as that of the original vaccine strain B. abortus 104M. With the intraperitoneal introduction of the strain culture to C ₅₇ Black / 6 mice and slaughter of animals followed by bacteriological examination of spleen ID _{50 of} strain KB 13/100 leaves 250-1000 microbial cells (p = 005).

 Immunogenic properties.

The strain culture introduced into guinea pigs under the skin at a dose of 1 • 10 ⁹ microbial cells protects 100% of the immunized animals from experimental infection with a virulent strain of brucella at a dose of 10-15 ID ₅₀ three months after vaccination.

 Stability of the main properties of the strain during passivation.

 The strain is stable in relation to the basic properties for 10 passages on solid and liquid nutrient media and 5 passages on guinea pigs (observation period).

 Strain B. abortus VGNKI N KB 13/100 is used in the manufacture of biological products for the diagnosis and prevention of animal brucellosis.

 Example 1

 The cultural properties of the vaccine strains of B. abortus KB 13/100, B. abortus 19 and B. abortus 104M are compared.

Two-day agar cultures of vaccine strain Brucella are suspended in sterile saline and plated in Petri dishes and tubes with solid and liquid nutrient media with and without 0.001% bis-triphenylhydrocarbinol oxalate. Crops are incubated at 37-38 ^o C for 4-5 days. The presence of brucella growth is taken into account and the number of colonies is counted. The research results are given in table. 1
The results of accounting for the growth of brucella, as follows from table 1, show that the vaccine strain B. abortus KB 13/100 is equally stably reproduced on media with bis-triphenylhydrocarbinol oxalate and without it. While cultures of strains 19 and 104M do not grow on media containing an extraneous microflora growth inhibitor.

 Example 2

 Strain B. abortus KB 13/100 was cultured on MPPGA containing 0.001% bis-triphenylhydrocarbinol oxalate. The brucella culture is washed off with buffered saline and an antigen is prepared for agglutination reactions and complement binding in a known manner.

At the same time, similar antigens are made from the bacterial strains of B. abortus 19 and 104M grown on MPPTTA without an inhibitor. Dilute antigens with phenolized saline to a concentration of 2 • 10 ⁹ microbial cells. Antigens are tested in the agglutination reaction (RA) and the complement binding reaction (CSC) with the national anti-brucellosis serum (1000 IU / cm ³ ).

 The results of determining the activity of antigens are presented in table. 2 and 3.

 The reaction results show that the antigen from the B. abortus KB 13/100 strain is superior in activity to the similar preparations from the B. abortus 19 and 104M strains.

 Example 3

The strain B. abortus KB 13/100 was cultured on MPAA, PHGA, and in MPHGB with the addition of 0.001% bis-triphenylhydrocarbinol oxalate. The B. abortus 19 and 104M strains were cultured on the same media without inhibitor. According to the known method, from the obtained bacterial mass of each of the three strains, live dry vaccines are prepared (three series of each strain). Immunogenicity of vaccines is tested in an active defense test in guinea pigs. Guinea pigs are immunized in doses of 1 • 10 ⁷ , 1 • 10 ⁸ , 1 • 10 ⁹ microbial cells. Vaccines are administered under the skin into the groin area. 90 days after immunization, the animals are infected with a standard culture of a control virulent strain of B. abortus 54 / M VGNKI at a dose of 20 ID ₅₀ . 32-35 days after infection, the animals are killed and bacteriological examination of the lymph nodes and internal organs is carried out according to the accepted method. The criterion for evaluating the effectiveness of the vaccine is the percentage of protection - the ratio of the number of animals not infected with brucellosis to the total number of guinea pigs in the group. An additional indicator of the effectiveness of vaccines is the infection index - the group average ratio of the number of selected brucella cultures to the number of studied objects, multiplied by 100. The results of determining the immunogenicity of anti-brucellosis vaccines are given in table. 4.

 As a result of the studies, it was found that the vaccine from strain B. abortus KB 13/100 is superior in specific efficacy to similar preparations from strains of B. abortus 19 and 104M.

 The use of B.abortus KB 13/100 strain for the manufacture of biological products for the diagnosis and prevention of animal brucellosis will significantly reduce the loss of raw materials, increase the sensitivity of diagnostic drugs and the specific effectiveness of anti-brucellosis vaccines.

Literature
1. Veterinary drugs. Directory. Ed. D.F. Osidze - M .: Kolos. 1981, p. 176-188.

 2. Shumilov K.V., Albertyan M.P., Klochkov A.A., Romakhov V.A. Cattle brucellosis vaccines. Veterinary Medicine - 1984, N12, p. 26-29.

Claims (1)

 The bacterial strain Brucella abortus VGNKI KV 13/100 DEPT used for the manufacture of biological preparations for the diagnosis and prevention of brucellosis in farm animals.

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