RU2812350C1 - Method of obtaining diagnostic serum against brucella in r-form - Google Patents

Abstract

FIELD: veterinary microbiology; immunology.

SUBSTANCE: method of obtaining diagnostic serum against Brucella in R-form, including obtaining a two-day culture from the B.abortus 16/4 strain in R-form with stable antigenic properties, increasing the bacterial mass and preparing a suspension without chemical exposure, by inactivation at 80°C for 60 minutes and three times washing from the medium with sterile saline solution under aseptic conditions. The finished preparation is inoculated into producing animals intravenously three times with an interval of 72 hours in increasing doses. Obtaining hyperimmune serum is carried out from 4 days after the last inoculation, and industrial bloodletting (total bleeding) is carried out from 15 to 42 days after the end of the immunization regimen. After bleeding, containers with blood are kept in a thermostat at 37°C for 3–4 hours to separate the whey, then they are placed in the refrigerator at 2–8°C for settling. Each batch of the resulting serum is subjected to ultrafiltration. Serum is tested for sterility, activity and specificity.

EFFECT: epidemic safety, the universality of the use of several modifications in RA as a control when assessing the humoral response and the ability to be used to determine the antigenic properties of isolated strains in the bacteriological diagnosis of biomaterial from animals immunized with non-agglutinogenic or weakly agglutinogenic vaccines or infected with Brucella in the R-form.

1 cl, 3 tbl, 2 ex

Description

The invention relates to veterinary microbiology and immunology and can be used as a control brucellosis serum in the production of test systems (kits) for the serological diagnosis of brucellosis in various modifications of the agglutination reaction (RA) for the differential assessment of the humoral response in animals immunized with non-agglutinogenic or weakly agglutinogenic vaccines or infected with Brucella in the R-form, and also as an agglutinating serum for the identification of isolated Brucella cultures.

Brucellosis is an infectious disease of animals and also poses a great danger to people, so improving the quality of diagnosis and its complete elimination is an urgent task for veterinary and medical science.

In Brucella, the phenomena of dissociation with the formation of (R-; S-; L-) forms, which have various cultural-morphological, antigenic, biological and other properties, have been described [1, 2].

Currently, in the laboratory diagnosis of brucellosis, diagnostic test systems that detect antibodies to Brucella in the S-form are widely used, therefore, taking into account the variability of the pathogen, the development of diagnostic drugs for identifying dissociated forms of the pathogen using immunological reactions remains relevant [3].

There is a known method for obtaining diagnostic serum against Brucella in L-form by transformation with streptomycin at a dose of 2.5-5.0 U/ml of the vaccine strain B.abortus 19 (S-form), stabilizing it on a solid nutrient medium with the addition of 10- 15% normal horse serum, obtaining antigen from a stable strain of B.abortus 19 in L-form, inactivation at a temperature of 85-90°C for 60 minutes, three-time intravenous hyperimmunization of rabbits in increasing doses of 60, 90, 120 billion m.k. . [4].

When creating the invention, the task was to develop a method for obtaining diagnostic serum suitable for the detection and identification of Brucella in the R-form, environmentally safe, ensuring universal application, not requiring additional economic costs, increasing the quantitative and qualitative indicators of the finished product.

The closest is the method of obtaining R-brucellosis serum in rabbits, including a single subcutaneous injection of a mixture of antigen (100 million CFU/ml of inactivated B.abortus 16/4 culture) with adjuvant MONTANIDE ^TM ISA 61 VG, a test blood draw on days 16-20 and from 21 to 35 days after hyperimmunization - three to four times blood sampling from each rabbit or total bleeding, obtaining serum, preserving the serum with sodium merthiolate [5].

The disadvantages of this method are: the use of the imported drug MONTANIDE ^TM ISA 61 VG as an adjuvant, manufactured by SEPPIC, France, the use of a preservative, which is a chemical compound, and the possibility of using the final product only in bacteriological diagnostics.

The technical result of the proposed method for obtaining diagnostic serum against Brucella in the R-form is the production of a drug from the B.abortus 16/4 strain with stable antigenic properties, increased efficiency due to the absence of additional expensive foreign components, environmental safety achieved through the use of inactivated biomass and sterilization of the resulting preparation mechanically without the use of chemicals, increasing the volume of the finished product due to the possibility of obtaining highly active brucellosis serum already from 4 days after the end of the immunization regimen, versatility of use in several modifications of the agglutination reaction when assessing the humoral response and studying the antigenic properties of isolated strains for bacteriological diagnostics biomaterial from animals immunized with non-agglutinogenic or weakly agglutinogenic vaccines or infected with Brucella in the R-form.

Rabbits were used as animal producers.

The technical solution is achieved by the fact that the method of obtaining diagnostic serum against Brucella in R-form on rabbits includes obtaining a two-day culture from the B.abortus 16/4 strain in R-form with stable antigenic properties, increasing the bacterial mass on matte flasks with a dense selective nutrient medium and preparing a suspension, which is inactivated at a temperature of 80°C for 60 minutes, washed three times from the medium under aseptic conditions by adding a sterile saline solution and centrifuging at 3000 rpm for 15-20 minutes. The finished preparation is inoculated intravenously into producing animals three times with an interval of 72 hours at an increasing dose of 60, 90, 120 billion CFU/ml, respectively. Obtaining hyperimmune serum is carried out from 4 days after the last inoculation, at the rate of 16-20 ml per 1 kg of live weight of the rabbit. Industrial bleeding (total bleeding) is optimally carried out from 15 to 42 days after the end of the immunization regimen.

After bleeding, containers with blood are left in a thermostat at 37°C for 3-4 hours to separate the serum, then placed in a refrigerator at 2-8°C to settle. Each batch of the resulting whey is subjected to ultrafiltration using a filter system with a pore diameter of no more than 0.22 microns. The serum is then tested for sterility, potency and specificity.

It must be sterile, not agglutinate in the reaction of RA with heterologous antigens from Brucella in the S-form and react positively with homologous R-antigens in titers of test tube RA - not lower than 1:80, plate - 1:20, with native R-cultures - not lower than 1:80.

The possibility of practical implementation of the proposed invention is confirmed by examples of specific implementation of the method using the full set of claimed features.

Example 1. The activity of each series of the obtained diagnostic R-serum was tested in a test tube agglutination reaction with a heterologous antigen (single antigen for RA and RSK “Test system for the diagnosis of brucellosis in RA, RSK, RDSC” FKP “Shchelkovo Biocombine”).

The specificity of each series of the obtained diagnostic R-serum was checked in tube and plate RA with antigen from Brucella R-strains, using the “Kit for differential serological diagnosis of brucellosis and monitoring the immune response of cattle immunized with a vaccine from the B.abortus 82 strain.” The research results are presented in Table 1.

Table 1 - Determination of activity and specificity of the obtained diagnostic R-serum of rabbits

Series No. Duration of the study (days) Study results (average titers for the group) RA test tube RA lamellar single
10-640 (IU) R
1:640 R
1:80 1 4 days - 1:160 1:80 2 7 days - 1:160 1:90 3 15 days - 1:640 1:60 4 21 days - 1:640 1:60 5 28 days - 1:560 1:60 6 35 days - 1:400 1:60 7 42 days - 1:400 1:60 8 49 days - 1:240 1:60

The highest activity of the obtained R-serum for all serological reactions with the R-antigen was noted on days 15-21 with a diagnostic titer (average for the group) in tube RA - 1:640, plate RA - 1:60.

When studying the specificity of the obtained series of diagnostic R-serum in serological reactions with the S-antigen, a negative result was noted in all cases.

Example 2. The effectiveness of using the obtained series of diagnostic serum for indication and differentiation of typical and modified forms of Brucella was studied using native cultures of vaccine strains with known antigenic properties - B.abortus 19 c.6 (S-form), B.abortus 16/4 ( R-form), B.abortus 82 p.3 (SR-form). The results are presented in Table 2.

Table 2 - Determination of the activity and specificity of diagnostic R-serum in lamellar RA with native cultures of vaccine strains of Brucella in (S-), (R-) and (SR-) form

Series No. Duration of the study (days) Study results (average titers for the group) B.abortus 19 p.6
(S-shape)
1:10 – 1:80 B.abortus16/4
(R-shape)
1:10 – 1:80 V.abortus 82 p.3
(SR form)
1:10 – 1:80 1 4 days - 1:80 1:20 2 7 days - 1:80 1:40 3 15 days - 1:80 1:35 4 21 days - 1:80 1:37.5 5 28 days - 1:80 1:37.5 6 35 days - 1:80 1:27.5 7 42 days - 1:80 1:27.5 8 49 days - 1:80 1:27.5

In all series of diagnostic serum, there was no agglutination with B.abortus 19 c.6 in the S-form, while in the reaction with B.abortus16/4 in the R-form, in all cases the formation of a persistent small agglutinate was observed in serum dilutions of 1:80 to "4 crosses". In reactions with B.abortus 82 p.3 (SR-form), the presence of small agglutinate in serum dilutions of 1:20-1:80 was also noted in all cases.

Example 3. To determine the effectiveness of using diagnostic R-serum in the study of cultures isolated from different animal species, Brucella strains, both reference and isolated from different animal species, were studied. The results are presented in Table 3.

Table 3 - Determination of the activity and specificity of diagnostic R-serum in RA on glass with native cultures of Brucella, reference strains and isolated from different animal species

No. Strain name Colony morphology Main owner
(kind of animal) Result of plate agglutination reaction on glass Dilution diagnostic
R-serums 1:5 1:10 1:20 1:40 1:80 Reference strains 1 B.abortus 544 S Cattle and other representatives of the genus Bovidae - - - - - 2 B.melitensis 16M S Sheep, goats - - - - - 3 B.suis 1330 S pigs - - - - - 4 B.ovis 63/290 R sheep # # # # # 5 B.canis 6/66 R dogs # # # # # Epizootic strains 6 V.abortus 1-15 S Cattle - - - - - 7 V.abortus 2-15 S Cattle - - - - - 8 V.abortus 3-15 S Cattle - - - - - 9 V.abortus 4-15 S Cattle - - - - - 10 V.abortus 9-71 S R Cattle # # - - - eleven V.abortus 120-77 S R Cattle # # # - - 12 B.abortus 111-89 S R Cattle # # # # - 13 B.abortus 147-89 S R Cattle # # # - - 14 B.ovis 246-90 R sheep # # # # # 15 B.ovis 2000-90 R sheep # # # # # 16 B.ovis 11-90 R sheep # # # # # 17 B.canis 1-06 R dogs # # # # # 18 B.canis 1-07 R dogs # # # # # 19 B.canis 1-08 R dogs # # # # # 20 B.canis 1-22 R dogs # # # # #

When determining the agglutinating properties of the studied Brucella cultures with previously studied antigenic properties in a complex of cultural-morphological, biochemical and other tests, the specificity of the lamellar agglutination reaction and the correspondence of the agglutinating properties of the strains to the morphological characteristics were noted.

Thus, diagnostic serum against Brucella in the R-form has strict specificity and high activity. The resulting drug can be used both in serological studies in various modifications of RA, in determining the humoral immune response to sensitization by Brucella in the R and SR form, and for differentiating Brucella cultures isolated during bacteriological examination, regardless of the type of animal.

The invention can be used in research and production laboratories dealing with the problems of animal brucellosis.

Literature

1. Degtyarenko L., Karlova M.Yu., Kalikin I.N. Diagnostic effectiveness of R-brucellosis antigens for brucellosis in cattle. Farm animal veterinary. 2015. No. 11. pp. 12-19;

2. Kosarev M.A., Fomin A.M., Safina G.M., Grigorieva S.A., Tukhvatullina L.A. Study of the cultural and morphological properties of Brucella abortus species, which are in varying degrees of dissociation. Veterinarian. 2018. No. 4. P. 14-18;

3. Manual for the diagnosis of brucellosis in animals. Approved by the Veterinary Department of the Ministry of Agriculture of the Russian Federation 09.29.2003 No. 13-5-02/0850;

4. Method for obtaining diagnostic serum against Brucella in L-form. Oshchepkov V.G., Gordienko L.N., Bratsev A.Yu. Patent RU2268748 C2, 01/27/2006. Bulletin No. 03. Application No. 2003121773/13 dated July 15, 2003;

5. Method for obtaining R-brucellosis serum in rabbits. Arakelyan P.K., Raznitsyna G.V., Yanchenko T.A., Dimov S.K., Dimova A.S. Patent RU 2659948 C1, 07/04/2018. Bulletin No. 9. Application No. 2017108572 dated March 14, 2017.

Claims (1)

A method for obtaining diagnostic serum against Brucella in R-form, including obtaining a culture from the R-strain B.abortus 16/4 with stable antigenic properties, preparing a bacterial suspension, hyperimmunization, bleeding, obtaining serum and testing the serum for sterility, activity and specificity, differing the fact that immunization is carried out intravenously three times in increasing doses discretely in time, and the suspension for injection is prepared without chemical exposure, by inactivation at 80 ° C for 60 minutes and three times washing from the medium with sterile saline under aseptic conditions, serum is obtained from 4 days after the last inoculation, bled from 15 to 42 days after the end of the immunization regimen, the resulting serum is subjected to ultrafiltration.