RU2263142C2 - Method for preparing l-subcultures of brucella abortus, strain i-206 - Google Patents

Abstract

FIELD: microbiology, immunology.

SUBSTANCE: proposed invention characterizes a method for preparing L-subcultures of Brucella abortus, strain I-206. Method involves passage of brucella of the strain Brucella abortus I-206 in nutrient medium comprising a liver infusion, normal equine serum, agar-agar. Sodium chloride, magnesium sulfate, sucrose and glycerol with addition of bicillinum-3 to the nutrient medium in increasing concentrations wherein these components are taken in the following ratio, wt.-%: agar-agar, 1.2; sodium chloride, 0.1; magnesium sulfate, 0.05; sucrose, 10; glycerol, 1.0; normal equine serum, 10; liver infusion, the balance, wherein bicillinum-3 is added to the nutrient medium in increasing concentrations from 10 to 9000 U/ml of medium. Method provides preparing L-subcultures of brucella in the final concentration of bicillinum-3 up to 2000 U/ml of medium wherein these microorganisms in a single intravenous administration in rabbits cause the production of specific noncomplete antibodies detected by the Kumbs reaction at titer value 1:400. At the final concentration of bicillinum-3 9000 U/ml of medium L-subcultures of brucella are prepared that in a single intravenous administration in rabbits cause the production of specific agglutinins detecting in the agglutination reaction at titer value 1:1600. Invention can be used in immunology in aim for preparing highly active and highly specific brucella L-forms serum.

EFFECT: improved preparing method.

2 tbl, 2 ex

Description

The invention relates to the field of medicine and veterinary medicine, in particular to microbiology and immunology, and can be used to obtain L-subcultures of B. abortus with high antigenic activity and specificity and used to prepare antigen in the production of diagnostic sera, as well as the determination of specific antibodies against L-form brucella in human and animal serum.

It is known that brucella in the L-form have weak immunogenic properties and cause the formation of specific antibodies (AT), which are detected in blood serum in the agglutination reaction (RA) in low titers not exceeding 1: 640; therefore, subcultures of brucella in the L-form with the goal of preparing antigen (AH) for immunization in obtaining highly active and highly specific sera is an urgent task.

A known method of producing L-subcultures of B. abortus using nutrient media, where the liver-glucose-glyceric medium is the most optimal basis for the nutrient medium (hepato-glucose-glycerol, Albimi and meat-peptone serum-dextrose). Using this medium as a basis, 14 variants of nutrient media were tested, which additionally included: agar-agar at a concentration of 0.14-0.35%, sucrose at concentrations of 10, 15 and 20%, magnesium sulfate 10, 15 and 20% , sodium chloride 0.1, 0.6, 1.5 and 3.0%, normal horse serum 20%. Penicillin was used as a transforming agent in doses of 0.15, 62, 125, 250, 1000, 5000, 10000 IU / ml of medium. However, in the study of the obtained L-subcultures of brucella with a specific anti-brucellosis serum labeled with fluorescein isocyanate, heterogeneity of the population of cultures, the presence of stable (diffuse luminescence) and unstable L-cells that retained the elements of the cell wall (causing luminescence) were noted (Ostrovskaya N.N., Vershilova P.A., Tolmacheva T.A. // Journal of Microbiol. - 1972. - No. 4. - S.108-112). Therefore, the use of these L-subcultures does not allow the preparation of a highly active and highly specific antigen for the production of immune antisera.

Closest to the proposed is a method for producing L-subcultures of B. abortus 149, 318, including the use of a medium containing semiliquid tryptic digestion of bovine hearts, 10-20% sucrose, 0.05-5% magnesium sulfate, 0.01-1 % sodium chloride, 15-25% horse serum, 1% glycerin, 0.01-0.03% nalidixic acid, and as a transforming agent, bicillin-3 from 50 to 20,000 IU / ml and glycine from 0.1% to 3 % However, the brucella L-subcultures obtained by this method are characterized by reduced, and sometimes completely lost antigenic properties. The antiserum obtained using the Brucella L-subculture data was not specific enough, since the initial bacterial S-forms were agglutinated at a dilution of 1: 320, and the homologous L-antigen was 1: 640, which indicates the preservation of pronounced antigenic bonds with the original parent strain (Bazikov I.A. L-transformation of brucella. / Epidemiology, microbiology and immunology of bacterial and viral infections. Abstracts of the regional scientific conference of young scientists, October 17-20, 1989, Rostov-on-Don, 1989, p.5- 8.) In addition, this method sufficient roads.

The aim of the invention is to obtain L-subcultures of brucella with high antigenicity and specificity, cheaper method.

This goal is achieved by repeated passivation of the strain B. abortus I-206 in S-form from the collection of live cultures of the Irkutsk Research Institute of Non-pedagogical Research, characterized by high antigenic activity and high resistance to penicillin antibiotics on a medium containing a transforming agent. The composition of the medium includes: hepatic infusion, agar-agar, sodium chloride, magnesium sulfate, sucrose, glycerin, normal horse serum in the following ratio, wt.%:

agar agar 1,2 sodium chloride 0.1 magnesium sulfate 0.05 sucrose 10 glycerol 1,0 normal horse serum 10 liver infusion rest

To obtain L-subcultures that are capable of inducing the production of incomplete antibodies in animals upon a single administration to animals, the initial B. abortus I-206 strain is passaged five times on nutrient medium to which bicillin-3 is added in increasing concentrations: 10-50-100-1000- 2000 IU / ml of medium, and to obtain L-subcultures that cause agglutinin production upon a single administration to animals, twelve-fold passaging on nutrient medium with bicillin-3 concentrations is carried out: 10-50-100-1000-2000-3000-4000-5000-6000 -7000-8000-9000 U / ml of medium.

Comparative analysis with the prototype showed that the proposed technical solution differs from the known one in that to obtain L-subcultures, multiple passaging of the strain B. abortus I-206 is used on a nutrient medium containing hepatic infusion, agar-agar, sodium chloride, magnesium sulfate, sucrose , glycerin, normal horse serum, in the following ratio of components, wt.%:

agar agar 1,2 sodium chloride 0.1 magnesium sulfate 0.05 sucrose 10 glycerol 1,0 normal horse serum 10 liver infusion rest

With repeated passaging, bicillin-3 is added to the nutrient medium in concentrations: 10-50-100-1000-2000-3000-4000-5000-6000-7000-8000-9000 IU / ml of medium.

Thus, the proposed technical solution meets the criteria of the invention of "novelty."

The analysis of patent and scientific and technical literature showed that the proposed method differs not only from the prototype, but also other technical solutions in this and related fields. So, the authors have not found a way to obtain L-subcultures of strain B. abortus I-206, including the proposed modes. Namely, these modes make it possible to achieve the goal - to obtain L-subcultures of B. abortus I-206 strain, which have high antigenic activity and specificity, which are used to prepare AH, which, when administered to rabbits, causes the production of highly active and highly specific ATs, and in the study of blood serum humans and animals detect AT against brucella in L-form. In addition, this method is cheaper, since it does not require special equipment and expensive reagents and can be carried out in ordinary laboratory conditions, as well as in the production of immunobiological preparations.

Thus, this method meets the criteria of "inventive step" and "industrial applicability".

The method is as follows:

A strain of B. abortus I-206 is taken and L-transformation is carried out by repeated passage on a nutrient medium containing liver infusion, agar-agar, sodium chloride, magnesium sulfate, sucrose, glycerin, normal horse serum, in the following ratio of components, wt.% agar-agar - 1.2, sodium chloride - 0.1, magnesium sulfate - 0.05, sucrose - 10, glycerin - 1.0, normal horse serum - 10, liver infusion - the rest. Bicillin-3 is added to the nutrient medium with repeated passaging in increasing concentrations: 10-50-100-1000-2000-3000-4000-5000-6000-7000-8000-9000 U / ml of medium. To obtain the L-subculture, which, when administered to animals once, induces the production of incomplete antibodies, the strain B. abortus I-206 is passaged on a nutrient medium to a concentration of bicillin-3 of 2000 IU / ml (fifth passage). To obtain an L-subculture that causes agglutinin production during a single administration to animals, passaging is carried out on nutrient medium to a concentration of bicillin-3 of 9000 U / ml of medium (12th passage). To obtain the bacterial mass of the L-subculture, 5 and 12 passages are sown on vials with culture medium and antibiotic content corresponding to the passage. After growing, the bacterial mass of L-subcultures is washed off with a physiological solution of pH 7.2-7.4, the concentration of microbial cells is determined by the industry standard sample of turbidity of bacterial suspensions (CCA 42-28-59-85) in 10 units To prepare the antigen, the bacterial mass is inactivated by the addition of 2.5% formalin and incubated for 24 hours. The antigen obtained in this way is monitored for specific sterility.

Changes in the biological properties of B. abortus I-206 I-206 L-subcultures of 5 and 12 passages compared to the original culture and reference brucella strains are shown in Table 1. During phase-contrast microscopy, smears of L-subcultures of brucella 5 and 12 passages show a polymorphic morphological picture, including the presence of a significant number of spherical and filamentous forms. The obtained L-subcultures do not lose their properties for two years (observation period). PCR results indicate that the studied microorganisms in S- and L-forms belong to the genus Brucella. As primers, oligonucleotides are used that limit the fragment of the chromosomal DNA of brucella of all types to 269 nucleotides. Primer sequences: Bru 1 (5 '- GCA GTC AGA CGT TGC STA TT - 3'); Bru 2 (5 '- GCT TCA GGT GTT CAG CCT T - 3'). A cell suspension of B. abortus I-206 (S-form) is used as a positive control, and distilled water as a negative control. The amplification program: 94 ° C - 35 sec, 52 ° C - 35 sec, 72 ° C - 35 sec, out of 40 cycles, is carried out on a multi-channel amplifier “MS-2” (CJSC DNA Technology. Moscow). To test the antigenic properties of B. abortus I-206 L-subcultures 5 and 12 passages, experimental animals (chinchilla rabbits) are used, which are injected with 1 ml of a suspension of living L-subculture cells at a dose of 10 ⁹ mk. After 7 days, the animals are bled and receive immune sera. The antigenic activity of the obtained L-subcultures is judged by the titer of specific AT in the blood serum of infected animals detected in the Coombs (RK) and RA reactions. The specificity of the obtained antigens from L-subcultures of B. abortus I-206 5 and 12 passages is checked in RA with different sera (table 2). As can be seen from the results presented in table 2, the high antigenic activity of L-subcultures of B. abortus I-206 strain 5 and 12 passages is evidenced by the fact that the antiserum obtained against passage L-subculture 5 contains incomplete ATs that give positive result in the Republic of Kazakhstan with L-subcultures of 5 and 12 passages in a titer of 1: 400. These same L-subcultures do not interact in RA. Antiserum obtained against passage 12 L-subculture contains agglutinins, which react positively in RA with 5 and 12 passage L-subcultures in a titer of 1: 1600. The high specificity of the obtained B. subcultures of B. abortus I-206 is indicated by positive results in RK (1: 400) and RA (1: 1600) with homologous antisera and negative in RA with sera: experimental against the initial B. abortus I-strain 206 in S-form; commercial: tularemia, cholera "O", cholera "Ogawa", yersiniosis 0: 9 and 0: 3. In this case, the initial S-culture of B. abortus I-206 reacted positively in RA with sera: experimental against the original strain B. abortus I-206 in S-form (1: 3200); commercial ones: tularemia, cholera "O", cholera "Ogawa" and yersiniosis 0: 3 (1:40), yersiniosis 0: 9 (1: 1600) and did not interact with experimental sera against L-subcultures of B. abortus I-206 5 and 12 passages.

Example 1

Take strain B. abortus I-206 in S-form and get the L-subculture of the fifth passage by repeated passaging on a nutrient medium containing liver infusion, agar-agar, sodium chloride, magnesium sulfate, sucrose, glycerin, normal horse serum in the following ratio components, wt.%: agar-agar - 1.2, sodium chloride - 0.1, magnesium sulfate - 0.05, sucrose - 10, glycerin - 1.0, normal horse serum - 10, liver infusion - the rest. Bicillin-3 is added to the nutrient medium in increasing concentrations: 10-50-100-1000-2000 U / ml of medium. To obtain the bacterial mass of the L-subculture of the fifth passage, operations are performed as described above.

Example 2

Take strain B. abortus I-206 in S-form and get the L-subculture of passage 12 by repeated passage on a nutrient medium containing hepatic infusion, agar-agar, sodium chloride, magnesium sulfate, sucrose, glycerin, normal horse serum in the following ratio components, wt.%: agar-agar - 1.2, sodium chloride - 0.1, magnesium sulfate - 0.05, sucrose - 10, glycerin - 1.0, normal horse serum - 10, liver infusion - the rest. Bicillin-3 is added to the nutrient medium in increasing concentrations: 10-50-100-1000-2000-3000-4000-5000-6000-7000-8000-9000 IU / ml of medium. To obtain the bacterial mass of the L-subculture of passage Brucella 12, operations are performed as described above.

The 5 and 12 passages of B. abortus I-206 strain obtained by this method have high antigenic activity, since when administered once to the rabbits, specific antibodies are detected that are detected in blood serum in the Republic of Kazakhstan in titers of 1: 400 (passage 5) and in PA - 1: 1600 (12 passage) and high specificity, as evidenced by the negative results in RA L-subcultures with sera: experimental against the original strain B. abortus I-206 in S-form; commercial: tularemia, cholera "O", cholera "Ogawa", yersiniosis 0: 9 and 0: 3. Inactivated AH prepared from the L-subcultures according to the proposed method is technologically advanced, since immunizing animals or performing serological reactions when specific ATs are detected in the blood serum of humans and animals does not require special measures to protect working personnel from brucellosis infection. Compared with the prototype, this method allows to obtain L-subcultures of brucella with higher antigenic activity and specificity, the introduction of which animals causes the formation of specific AT in higher titers in RA 1: 1600 and in RK 1: 400, in the prototype in PA - 1 : 640, while the antisera obtained against the proposed L-subcultures of B. abortus I-206 do not interact in RA with the original strain in the S-form, in the prototype - in the titer of 1: 320. The high specificity of the proposed L-subcultures is evidenced by the negative results of RA with sera that cross-react with brucellas in the S-form, commercial: tularemia, cholera "O", cholera "Ogawa", yersiniosis 0: 9 and 0: 3. Antigen prepared from L-subcultures of B. abortus I-206 5 and 12 passages is used to obtain highly active and highly specific diagnostic serum for detection of brucella in L-form, as well as to determine specific AT for brucella in L-form in human blood serum and animals.

In addition, the proposed method for producing L-subcultures of brucella compared to the prototype is cheaper.

Table 1
Biological properties of brucella in S- and L-forms Strains Lysis phage TB Dissociation Bacteriostatic properties Agglutability Production of hydrogen sulfide (mm) Urease activity (on Christensen's medium) reaction with tripaflavin thermal precipitation reaction thionine magenta polyvalent serum monospecific serums 1 day 2 days 3 days 20 minutes 2 hours 5:00 24 hours 1:25 thousand 1:50 thousand 1: 100 thousand 1:50 thousand 1: 100 thousand A M B.m.16M - - - + + + + + 1: 1600 - 1: 160 3 3 3 - Words Words +++ B.a. 544 + - - - - - + + 1: 1600 1:40 - Words Words 2 - - - - B.suis 1330 - - - + + + - - 1: 1600 1:40 - 6 5 5 + ++ +++ ++++ V.a.I-206
S-shape + - - - - - + + 1: 1600 1:80 - 1 Words Words - - Words +++ V.a.I-206
L-shape (5 passage) - + + + + + + + * 1: 100 - - - - - + ++ +++ ++++ V.a.I-206
L-shape (12 passage) - + + + + + + + * 1: 100 - - - - - - ± ++ +++ Note:
- the result of the interaction is not typical (there is no umbrella, the supernatant is cloudy, the formation of cords)
B.a. - B. abortus
Bm - B. melitensis
Words - trace amounts
A - antiabortus
M - anti-melitensis

table 2
The specificity of brucellosis S and L antigens in agglutination and Coombs reactions Serum Antigens B. abortus I-206 in L-shape (5 passage) B. abortus I-206 in L-shape (12 passage) B. abortus I-206 in S-form Experimental against B. abortus I-206 in S-form Neg. * Neg. * 1: 3200 * Experimental against B. abortus I-206 in L-form (5 passage) 1: 400 **
Neg. * 1: 400 **
Neg. * Neg. **
Neg. * Experimental against B. abortus I-206 in L-form (12 passage) 1: 1600 * 1: 1600 * Neg. * Commercial tularemia Neg. * Neg. * 1: 40 * Commercial cholera "O" Neg. * Neg. * 1: 40 * Commercial cholera "Ogawa" Neg. * Neg. * 1: 40 * Commercial Yersinious 0: 9 Neg. * Neg. * 1: 1600 * Commercial Yersinious 0: 3 ± * Neg. * 1: 40 * Note:
* result of agglutination reaction
** Coombs reaction result

Claims (1)

A method of obtaining B. subcultures of B. abortus, including the passage of brucella in a nutrient medium containing sodium chloride, magnesium sulfate, sucrose, glycerin, normal horse serum and bicillin-3 in increasing concentrations, characterized in that they use B. abortus I-206 strain and the nutrient medium additionally contains hepatic infusion and agar-agar in the following ratio of components, wt.%: Agar agar 1,2 Sodium chloride 0.1 Magnesium sulfate 0.05 Sucrose 10 Glycerol 1,0 Normal horse serum 10 Liver infusion Rest and bicillin-3 is added to the nutrient medium in increasing concentrations from 10 to 9000 U / ml of medium.