RU2486916C2 - Method for producing brucellous l-antigen - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to veterinary microbiology, immunology, biotechnology, namely to technology of an antigen for diagnosing brucellosis. The method for producing brucellous L-antigen is implemented as follows. The strain Brucella abortus 19 is cultured in the L-form on a solid nutrient medium prepared of meat-peptone liver glucose-glycerol agar (1.3-1.5% agar) with added 10-15% normal horse serum and streptomycin in a dose of 2.5-5.0 Units/ml at 37-38°C for 4-5 days. Then the prepared L-form culture is emulsified by 0.5% phenolised physiological saline; the suspension is inactivated at temperature 85-90°C for 60 minutes, centrifuged at 3000-5000 rpm for 15-20 minutes; the brucella concentration is specified at 50-60 bln microbial cells; the prepared antigen is titred, standartised by specificity and activity. The latter is used for the purpose of an agglutination test in serological diagnosis of brucellosis. The invention enables higher effectiveness and reliability of diagnosing brucellosis by 20-25%.

EFFECT: invention may be used for diagnosing brucellosis in carrier animals with persistent changed L-forms of the agent.

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Description

The invention relates to veterinary microbiology, immunology, biotechnology, and in particular to a technology for producing antigen for the diagnosis of brucellosis.

Brucella, like many other pathogens of infectious diseases, can exist in nature in S-, R- and L-forms, which differ from each other in morphological and biological properties, the degree of pathogenicity and specificity of antigenic determinants [1, 2, 5, 6, 10, 11].

Existing serological diagnostic methods allow the identification and identification of brucellosis caused by persistence of the pathogen in a typical (S-) or dissociated (R-) form, and animals infected with altered L-forms of brucella are not detected.

There is a method of producing an antigen for the diagnosis of brucellosis [7], according to which the vaccine strain B. abortus 19 is grown in solid nutrient medium, the grown culture is washed off with citrate buffer with a pH of 5.3-5.5, bacterial cells are inactivated by heating, and suspension is suspended by 12% sodium chloride solution to a final concentration of 2 × 10 ¹⁰ CFU / ml; methylene blue dye and phenol preservative are added. The resulting antigen is used in Wright and Heddelson agglutination reactions to diagnose brucellosis. However, this diagnosticum does not allow to identify animals infected with altered L-forms of brucella.

A known method of manufacturing a single brucellosis antigen for RA, RSK and RDSK, including the cultivation of strain 19, concentration and purification of the grown Bakmass using 0.5% phenolized saline and inactivation of the antigen [8]. However, this diagnosticum also does not allow the detection of animals infected with altered L-forms of brucella.

The known "Method for the production of diagnostic serum against brucella in the L-form", to obtain which used cultures from strain B. abortus 19 in the L-form by transforming the original typical culture of the vaccine strain Brucella abortus 19 (S-form) by cultivation in a solid nutrient medium, prepared on the basis of meat-peptone liver-glucose-glycerol agar (1.3-1.5%) (MPPGTA) with the addition of normal horse serum 10-15% and streptomycin in a dose of 2.5-5.0 U / ml of medium 37-38 ° C for 4-5 days, the antigen is obtained by inactivation of the obtained culture by heating at a temperature of 85-90 ° C for 60 min, and hyperimmunization of rabbits is carried out by triple intravenous administration of antigen in increasing doses. [9]. However, a stable L-culture was used only to obtain diagnostic serum against brucella, for bacteriological diagnosis of brucellosis.

The technical result of the invention is to increase the specificity of diagnostics by obtaining brucellosis L-antigen: in a complex of serological studies in the diagnosis of brucellosis.

The claimed technical result is achieved by the fact that the brucellosis antigen is obtained as follows: the Brucella abortus 19 strain in L-form is cultured on a solid nutrient medium prepared on the basis of meat-peptone liver glucose-glycerol agar (1.3-1.5%) with the addition of 10-15% of normal horse serum and streptomycin at a dose of 2.5-5.0 U / ml, at 37-38 ° C for 4-5 days, the culture obtained in the L-form is emulsified with 0.5% phenolized saline solution, inactivate at 85-90 ° C for 60 minutes, the suspension is centrifuged at 3000-5000 rpm for 15-20 minutes and establish the concentration of brucella 50-60 billion MK, the obtained antigen is titrated, standardized for specificity and activity and used for the agglutination reaction in the serological diagnosis of brucellosis.

A distinctive feature of the proposed method is that the preparation of the antigen is based on the use of a stable culture of Brucella abortus 19 in L-form for serological diagnosis of brucellosis.

The proposed brucellosis L-antigen has strict specificity for S, R, N-serum, diagnostic activity, can be used in a complex of serological studies in the diagnosis of brucellosis.

Example 1. To obtain brucellosis L-antigen using a stable culture from strain B. abortus 19 in L-form, obtained by transforming the original, typical culture of the vaccine strain B. abortus 19 (S-form) by cultivation in a solid nutrient medium prepared based on meat-peptone hepatic glucose-glycerol agar (1.3-1.5% agar) (MPPGTA) with the addition of normal horse serum 10-15% and streptomycin at a dose of 2.5-5.0 U / ml at 37- 38 ° C for 4-5 days. As a result of repeated studies, it was found that the formation of L-forms occurs when the initial cell (S-form) of brucella streptomycin in a dose of 2.5-5.0 U / ml of medium. A lower concentration of the antibiotic did not cause the formation of L-forms of brucella, and a higher concentration led to stunted growth and often death of the culture [1].

Table 1. Obtaining L-culture of brucella in a stable state in vitro experiments Study strain Dose of streptomycin (U / ml medium) 0.1 0.25 0.5 1,0 2,5 5,0 10.0 20,0 40,0 B. abortus 19 S S S S L L O O O

Note: S - typical S-forms of the pathogen; L - modified L forms of the pathogen; O - lack of growth.

When sowing the obtained L-culture on solid nutrient media, round-shaped colonies are formed, flattened, smooth with even edges, whitish-gray in color. On the broth, growth is accompanied by uniform turbidity of the medium, followed by enlightenment and the formation of an abundant loose white precipitate at the bottom of the tube.

Microscopy of native smears obtained L-culture was a gram-positive giant cells of an ellipsoid shape (ovoids) with a size of 2.5-3.0 × 5.0-6.0 μm.

L-form of B. abortus 19 to a large extent loses a rigid cell wall. It is agglutinated by L- and not agglutinated by S- and R-brucellosis sera. It gives a positive reaction of thermoagglutination and a test with tripaflavin. Does not emit hydrogen sulfide. It grows well on media with thionine and fuchsin. It is characterized by slightly virulent properties [9].

The method of producing brucellosis L-antigen is as follows: take a culture from Brucella abortus 19 strain in L-form, cultivate on meat-peptone hepatic glucose-glycerol agar (1.3-1.5% agar) with normal horse serum 10 -15% and streptomycin in a dose of 2.5-5.0 U / ml of medium at 37-38 ° C for 4-5 days. The grown back mass is washed off from a dense nutrient medium and emulsified with 0.5% phenolized saline solution to obtain a homogeneous consistency, which is inactivated at a temperature of 85-90 ° C for 60 minutes, then centrifuged at 3000-5000 rpm for 15-20 minutes . The resulting concentration of microbial cells is high, and it is brought to a concentration of 50-60 billion mk. (according to the turbidity standard of the GISK named after L.A. Tarasovich) in 0.5% phenolized saline solution.

The resulting antigen is titrated in dilutions 1:10, 1:20, 1:40, 1:60, 1:75. The antigen is recognized as suitable for diagnostic purposes at a 1:20 dilution, which was tested on homologous and heterologous sera. The obtained antigen is used for the agglutination reaction in the serological diagnosis of brucellosis.

Example 2. The specificity of the L-antigen was checked in the corresponding serological reactions using S- and R-brucellosis sera from commercial diagnostic kits and negative (N-) blood serum of healthy animals. The antigen for the agglutination reaction (RA) from brucella in the L-form gives a positive reaction only with L-brucellosis sera (Tables 2, 3, 4).

table 2 The results of the agglutination reaction (RA) of L-brucellosis antigen with brucellosis (S-, R-) and negative (N-) sera Dilution of L-brucellosis antigen Names and breeding of brucellosis sera S- (1:10) R- (1:10) N- (1:10) L- (1:10) 1:10 neg neg neg ++++ 1:20 neg neg neg ++++ 1:40 neg neg neg ++++ 1:60 neg neg neg ++ 1:75 neg neg neg -

The table shows that in cross-reactions with heterologous (S- and R-) sera, negative results were obtained, and with homologous (L-) sera, positive results were obtained.

Thus, the proposed brucellosis L-antigen has strict specificity for S, R and N sera and diagnostic activity. The working concentration of microbial cells of L-antigen is 50-60 billion.

Example 3. A production test of brucellosis L-antigen was carried out on a reindeer herd in herds with different epizootic characteristics for brucellosis with a total number of 8232, including 1944 heads for brucellosis dysfunctional.

The specificity of the L antigen was determined on the number of brucellosis-free reindeer herds (Table 3).

The data presented confirm that the antigen obtained from L-cultures has specificity and exhibit diagnostic activity in the agglutination (PA) reaction with serums that do not contain S, R, L antibodies.

In the foci of brucellosis infection, as shown by our previous bacteriological studies [3, 4], animals are carriers of not only S, -R-, but also L-forms of the pathogen, which are not detected by known S, -R-antigenic diagnostics, and in addition identified by the proposed L-antigen (table 4).

From the table it can be seen that out of 1944 examined animals, 134 reactive animals were identified, which made up 6.9% of the total number of animals examined, including 79.1% animals (106 animals) were identified with the known S-antigen, and the proposed L- the antigen allowed additional detection of 20.9% (28 animals) of brucellonimus animals with the persistence of the pathogen in L-form.

Thus, the proposed brucellosis L-antigen used in the agglutination reaction (RA) has strict specificity for S-, R- and N-serums, diagnostic activity, increases the efficiency and reliability of the diagnosis of brucellosis by 20-25%. It can be used to diagnose brucellosis in brucelloniferous animals with persistence of altered L-forms of the pathogen.

Literature

1. Bazikov I.A., Bondarenko A.N. Ultrastructural organization of L-forms of brucella and revertant cultures // ZhMEI. - 1991. No. 1. - S.17-20.

2. Bakulov I.A., Zelentsova T.Ya. The problem of L-forms of bacteria in veterinary medicine // Veterinary medicine. - 1980. No. 10. - P.23.

3. Gordienko L.N., Brattsev A.Yu., Bronnikov V.S., Popova T.G. Variability of the causative agent of brucellosis in the body of cattle // Actual problems of veterinary medicine in modern conditions: Mater. All-Russian scientific. - practical conf. - Penza, 2003 .-- S.15-17.

4. Gordienko L.N. Properties of altered forms of brucella. isolated from reindeer // Agriculture of the North at the turn of the millennium: Sat. scientific tr part 1. - Magadan, 2004.S. 236-239.

5. Zykin L.F., Vasiliev D.A. L-forms of pathogens of zooanthroponosis. - Ulyanovsk, 2000.68 s.

6. Kachesova L.M. The results of the study on unstable (reversible) L-brucellosis bacteriosis in cattle in areas of safe and dysfunctional for brucellosis // Sat. scientific tr LVI, 1982. No. 72. - 56-60.

7. Patent RU 2133470 C, G01N 33/569, A61K 39/10, publ. 07/20/1999.

8. Patent RU 2085212 C, A61K 39/10, G01N 33/569, C12N 1/20, publ. 07/20/1999.

9. Patent RU No. 2268748, C2, A61K 39/40, C12N 1/20, G01N 33/531, publ. 01/27/2006.

10. Rementsova M.M., Nifantiev V.M. The value of the L-forms of brucella in the epizootic process // Actual issues of the prevention of brucellosis and the organization of honey. patient care: thes. doc. All-Union Conference. - Moscow. 1989. - S. 93-94.

11. Trilenko P.A. MVB and L-forms of brucella // LVI. - 19 71. Issue. Number 32. - S. 35.

Claims (1)

A method for producing brucellosis L-antigen, comprising cultivating a strain of Brucella abortus 19 in L-form on a solid nutrient medium prepared on the basis of meat-peptone liver glucose-glycerol agar (1.3-1.5% agar) with the addition of 10-15% normal horse serum and streptomycin in a dose of 2.5-5.0 U / ml at 37-38 ° C for 4-5 days, inactivation at a temperature of 85-90 ° C for 60 minutes, characterized in that obtained in L -form culture is emulsified with 0.5% phenolized saline solution, centrifuged at 3000-5000 rpm for 15-20 m in and establish the concentration of brucella 50-60 billion MK, the resulting antigen is titrated, standardized for specificity and activity and used for the agglutination reaction in the serological diagnosis of brucellosis.