Method of obtaining of brucellous l-antigen

FIELD: biotechnologies.

SUBSTANCE: method of obtaining of brucellous L-antigen is performed as follows. The strain Brucella abortus 19 is cultivated in the L-form in the dense nutrient medium prepared on the basis of meat-peptonic hepatic glucose-glyceric agar (1.3-1.5%) with adding of 10-15% normal horse Serum at the temperature 20-22°C within 2-3 days. The culture, obtained in the L-form, is emulsified with 0.5% phenolised normal saline solution, inactivated at the temperature 85-90°C within 60 minutes. The suspension is centrifuged at 3000-5000 rpm within 15-20 minutes and the concentration of brucellas is established at the level 50-60 billion m.k. The obtained antigen is titrated, standardised by specificity and activity. It is used for agglutination test in serum diagnostics of brucellosis.

EFFECT: invention allows to minimise the time of antigen preparation and to increase the yield of bacterial mass.

4 tbl, 3 ex

 

The invention relates to veterinary Microbiology, immunology, biotechnology, in particular to a technology for production of antigen for the diagnosis of brucellosis.

Diagnostics and specific prophylaxis of brucellosis imperfect, due to the variety of forms of the organism and its ability to intracellular parasitic, which leads to the unavailability of disease existing diagnostic methods, which are mainly based on the use of antigens from the S-forms of Brucella (Counsel for the diagnosis of brucellosis, 2004) [1].

Each existing form (S - R - L-) of the microorganism when sown on nutrient medium has its own characteristics, which are clearly defined only for S-shape (Counsel for the diagnosis of brucellosis, 2004) [1].

A method of obtaining L-subcultures of B. abortus (Bazikov I. A. "L-transformation Brucella", 1989), including the use of the environment containing the basis of semi trypticase prewar bovine hearts, 10-20% sucrose, 0.05 to 5% of magnesium sulfate, 0.01 to 1% sodium chloride, 15-25% horse serum, 1% glycerol, 0.01 to 0.03% of nalidixic acid, as well as transforming Agusta bicillin-3 from 50 to 20,000 IU /ml and glycerol from 0.1%to 3%. However, obtained by the method of L-subculture Brucella have reduced and sometimes completely lost antigenic properties. Anticavity, poluchena is using data L-subcultures Brucella, was not sufficiently specific, as they agglutinable source of bacterial S-shape in a dilution of 1:320. Anticavity was proposed for bacteriological diagnosis of brucellosis [2].

A method of obtaining L-subcultures - "Method for L-subcultures strain brucella abortus And-206" (patent No. 2263142 from 2003.07.14). The method involves the passage of Brucella strains of Brucella abortus And 206 on a nutrient medium containing: agar-agar - 1,2; sodium chloride - 0,1; magnesium sulfate - 0,05; sucrose - 10; glycerol - 1,0; normal horse serum - 10; hepatic infusion - else, where bicillin-3 added to the culture medium in increasing concentrations from 10 to 9000 IU/ml of medium. This method allows you to get L-subculture Brucella at a final concentration of bicillin-3 2000 IU/ml of medium get L-subculture Brucella, which after a single intravenous administration to rabbits cause the production of specific agglutinins detected in agglutination reactions in titer of 1:1600 [3]. Cultivation of Brucella L-form occurs with the use of bicillin-3 in increasing concentrations from 10 to 9000 IU/ml of medium is a long process. And getting even a small amount of bacterial mass was used to obtain hyperimmune serum, which was used in the bacteriological diagnosis of brucellosis.

The closest technical solution t is aetsa a method of obtaining diagnostic serum against Brucella in an L-shape, to obtain which used culture of the strain of C. abortus 19 in an L-shape by transforming the original typical culture vaccine strain Brucella abortus 19 (S-form) cultivation on solid nutrient medium prepared based on meat-peptone hepatic glucose-glycerol agar (1,3-1,5%) (MPGGE) with the addition of normal horse serum 10-15% and streptomycin at a dose of 2.5 to 5.0 IU/ml at 37-38°C for 4-5 days, the antigen is produced by inactivation of the obtained culture by heating at a temperature of 85-90°C for 60 min and three times intravenously administered to rabbits in increasing doses with an interval of 72 hours [4]. The disadvantage of this method is the long process of preparation of the antigen, the purpose of this invention has been receiving a stable L-culture, and used the antibiotic streptomycin, which retards the growth of microorganisms and transition it into the original S-shape, the output of the bacterial mass is unimportant, since the introduction of culture into the body of an experimental animal antibodies (serum), which is used for bacteriological diagnosis of brucellosis.

The technical result of the invention is to reduce timing brucellosis L-antigen, increased output of finished products, the use of antigen in complex serological studies when diagnostics is brucellosis, namely agglutination reaction (RA) to identify additional infected animals with latent brucellosis.

The claimed technical result is achieved in that a method of obtaining brucellosis L-antigen includes the cultivation of Brucella abortus strain 19 in an L-shape on the solid nutrient medium prepared based on meat-peptone hepatic glucose-glycerol agar (1,3-1,5%) with the addition of 10-15% normal horse serum inactivation suspension at a temperature of 85-90°C for 60 minutes, the cultivation is carried out at 20-22°C for 2-3 days, received in an L-shape culture emuleret 0,5% fenrisians saline solution, centrifuged at 3000-5000 rpm for 15-20 minutes and set the concentration of Brucella 50-60 billion, M. K., the resulting antigen is titrated 1:5, 1:10, 1:20, 1:40, standardizes on specificity and activity, used for agglutination reactions in serological diagnosis of brucellosis. The antigen recognized suitable for diagnostic purposes in a dilution of 1:20.

The proposed method for the preparation of brucellosis L-antigen 2 times reduces the time of manufacture and increases the yield of bacterial mass to prepare antigen 3 times. The antigen used in the complex serological tests in the diagnosis of brucellosis, namely in PA to identify additional infected animals with l is a competent form of brucellosis.

The invention is characterized by the following examples.

Example 1. Culture of Brucella abortus strain 19 in an L-shape inoculated in tubes with nutrient dense environment, based on meat-peptone hepatic glucose-glycerol agar (1,3-1)5%) with the addition of 10-15% normal horse serum and streptomycin at a dose of 2.5 to 5.0 U/ml, at 37-38°C for 4-5 days. Microbial suspension of Brucella abortus 19 L wash saline solution of 0.85% NaCl), prepare one billionth mixture (according to the turbidity standard gisk named after. L. A. Tarasevich). Then by the method of successive dilutions choose a concentration to 1 ml was 100 m K. [5].

To study take 7 temperature conditions: 18°C, 20°C, 22°C, 25°C, 30°C, 35°C, 37°C. For each mode serves 4 Petri dishes with the environment, based on meat-peptone hepatic glucose-glycerol agar (1,3-1,5%) with the addition of 10-15% normal horse serum. In the first option in the environment add streptomycin, in the second variant without streptomycin. And in each Petri dish contribute 100 M. K. culture Brucella abortus 19 L, which stand in different temperature regimes, the growth of culture Brucella abortus 19 L check on Petri dishes visually daily for 5 days (table.1).

In different temperature regimes in environments with streptomycin and without culture C. abortus 19 L on Petri dishes grew up differently. When temperature is re 20°C, 22°C on media without antibiotics for 2-3 days grew colonies in 3 times more than with streptomycin. At 37°C for 5 days has grown colonies of the same number with streptomycin and without it, the number of colonies was less than in the first example.

In the proposed method of obtaining brucellosis L-antigen for 2-3 days at a temperature of 20-22°C was observed maximum growth of bacterial mass. Cultivation of B. abortus 19 L at a temperature of 20-22°C 2 times reduces the time of preparation of antigen and 3 times the output of the bacterial mass.

Table 1
The dependence of the growth culture of B. abortus 19 L from the temperature regime for 5 days
TemperatureDay (1-5)Medium with streptomycin, Petri-dishMedium without antibiotic Petri-dish
18°C12341234
12522201852535549
22830323054555855
35052505272757068
45253525381828382
56765535483828485
12221222175747576
22428253010099101100
347445052151150149150
450495153200201199271
566625354270269271270
22°C12321202176757477
225272427135140138135
346424847155158158156
451475353200205206210
567686059269272 273270
25°C12624242370666965
22931323074697273
35051505288898586
455545355104109107108
566656059150 145146148
30°C14546424346485051
26067666569696870
37872717489919394
484838881100106ON116
590898885 120118117118
35°C14647485029333130
26870667577747372
38582808693989296
49597949499100101100
5106107109 108111106113110
37°C14849505128323033
26969687075747677
38989858499989897
49998979810099100101
5109110 111110110108112110

Example 2. The specificity of L-antigen was tested in the relevant serological reactions with the use of S - and R-brucellosis sera from commercial diagnostic kits and negative (N-) of the blood serum of healthy animals. Antigen for agglutination reaction (RA) of Brucella in an L-shape gives a positive reaction only with L-Brucella serum (table.2, 3, 4).

Table 2
The results of the reaction of agglutination (PA) L-Brucella antigen with brucellosis (S,R) and negative (N-) sera
Breeding L-Brucella antigenNames and cultivation of Brucellasera
S-(1:10)R-(1:10)N-(1:10)L-(l:10)
1:5negnegneg++
1:10 negnegneg+++
1:20negnegneg++++
1:40negnegneg++++

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

From the above data it follows that the proposed brucellosis L - antigen, has a stringent specificity in relation to S, R and N sera and diagnostic activity. The working concentration of microbial cells L-antigen 50-60 billion

Example 3. Production testing of brucellosis L-antigen was performed on the population of deer in herds with different epidemic response of brucellosis total number 4400 goals, including affected with brucellosis -1005 head.

The specificity of L-antigen was determined on the reindeer head with a good brucellosis (table.3).

Total
Table 3
The results of serological tests for brucellosis in reindeer in prosperous stud
Year researchThe district of Yamal-Nenets Autonomous districtThe studied animals (animals)Of them respond positivelyWith antigens
SRL
goal.%
2010Yamal300000---
2011Yamal100000---
2012Yamal40000
440000

These data confirm that the antigen is derived from L-cultures, has specificity and shows diagnostic activity in the reaction of agglutination (PA) with sera, nesteriak S-, R-, L-antibodies.

In foci of brucellosis infection animals are native not only S-and L-forms of the pathogen that are not registered commercial S-antigen-based assays, and additionally identifies the proposed L-antigen (table.4).

Table 4
The results of serological tests for brucellosis in reindeer from disadvantaged stud
Year researchThe district of Yamal-Nenets Autonomous districtThe studied animals (animals)Of them respond positivelyWith antigens
SL
goals %
2008Nadym18421,02-
2009Taz37930,83-
2010Nadym3154831,52226
2011Nadym12743,122
Total100557-2928
% of investigated-2,82,7

The table shows that of the 1005 and the following goals identified positively reactive - 57 goals, which amounted to 5.5% of the total number of investigated animals, including the well-known S - antigen identified 2,8% of the animals (29 goals), and the proposed L-antigen allows to identify 2,7% (28 goals) animal brutselloosile with persistence of the pathogen in the L-form.

Thus, the proposed brucellosis L-antigen used in the reaction of agglutination (PA), has a strict specificity in relation to the S-, R - and N-sera, diagnostic activity, increases the efficiency and reliability of the diagnosis of brucellosis by 50.0%. Can be used for the diagnosis of brucellosis in animals-brutselloosile with persistence of the modified L-forms of the pathogen.

The invention allows to reduce in 2 times the time of preparation of antigen due to a change in temperature during the cultivation of Brucella abortus 19 in an L-shape and to increase the output of the bacterial mass in 3 times, the resulting antigen can be used in PA, in the complex serological tests for brucellosis to identify additional infected animals with latent infection that will improve the efficiency and reliability of the diagnosis of brucellosis by 50%.

LITERATURE

1. Counsel for the diagnosis of brucellosis in animals. - M., 2004. - 62 S.

2. Bazikov I. A. L-transformation Brucella / Epidemiology, Microbiology and immunology bacterial and viral is Pecci: abstracts. regional scientific. proc. young. scientists, 17-20 October 1989, Rostov-on-don, 1989, pp. 5-8.

3. Patent RU No. 2263142 C2, 7C12N 1/20, C12N 1/20, C12R 1:00 "How to get L-subcultures strain Brucellaabortus And -- 206", published 2005.10.27.

4. Patent RU No. 2268748 C2, A61K 39/40, C12N 1/20, G01N 33/531, publ. 27.01.2006.

5. Alton J., Jones, L. M. Methods of laboratory studies on brucellosis / FAO / who, Geneva, 1968. - 86 C.

The method of obtaining brucellosis L-antigen, including the cultivation of Brucella abortus strain 19 in an L-shape on the solid nutrient medium prepared based on meat-peptone hepatic glucose-glycerol agar (1,3-1,5% agar) supplemented with 10-15% normal horse serum, inactivation of the suspension at a temperature of 85-90°C for 60 minutes, characterized in that the cultivation is carried out at 20-22°C for 2-3 days, received in an L-shape culture emuleret 0,5% fenrisians saline solution, centrifuged at 3000-5000 rpm for 15-20 minutes and set the concentration of Brucella 50-60 billion, M. K., the resulting antigen is titrated standardise on the specificity and activity and is used for agglutination reactions in serological diagnosis of brucellosis.



 

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18 cl, 8 tbl, 4 ex

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.

4 tbl, 3 ex

FIELD: biotechnology, vaccines.

SUBSTANCE: vaccine comprises bacterial mass of Pasteurella multocida of serovariants A, B and D, Haemophilus pleuropneumonia of serogroups 1 and 2, and streptococcus of serogroups C and R, and also lysate-anigens of salmonellae Salmonella cholerae - suis, strain № 370 and Salmonella typhimurium № 415 mixed in the definite concentration. Vaccine elicits the high immunogenicity and provides the protection of pigs against infectious pneumonia of bacterial etiology and salmonellosis.

EFFECT: valuable veterinary properties of vaccine.

3 cl, 1 tbl, 5 ex

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