EA009817B1 - Method for detecting tuberculosis infection - Google Patents

Abstract

A method for detecting tuberculosis infection is proposed by a biomaterial incubation in a growth stimulator of VKG, sowing onto a nutrient medium VKG and cultivation thereof. The method is characterized in that after cultivation the grown culture is mixed with antiserum to antigens of mammal's tuberculosis stimulus and negative reference serum for providing agglutination test and by forming representative agglutinate it can be judged that the culture belongs to the mammal's tuberculosis stimulus, wherein the antiserum is obtained by injecting animals not heated disintegrated mycobacteria with subsequent adsorption thereof by cultures of atypical mycobacteria and banal microflora grown on the nutrient medium of VKG.

Description

The invention relates to the field of medicine and veterinary medicine, and in particular to methods of microbiological diagnosis of tuberculosis.

For the diagnosis of tuberculosis, as a rule, methods are used, including the selection and seeding of the biomaterial on nutrient media, cultivation and identification of grown cultures by morphological, cultural, biochemical, antigenic, pathogenic and other properties (I.R. Dorozhkova, S.A. Popov, VA Puzanov, LP Martynova, Microbiological studies in the detection, diagnosis and treatment of tuberculosis. Part 3. M., 2000, 119 p .; Microbiological diagnosis of tuberculosis and mycobacteriosis, in the book: “Tuberculosis.” Ed. A.G. Khomenko, M., Medicine, 1996; “Having started ix TB diagnosis of animals ", approved. GID Gosagroprom g .; 25.02.1986 USSR patent № 2193068 C2. VI, publ. 20.11.2002, at Bul. № 32).

The disadvantages of these methods are their low sensitivity, time consuming (up to 3 months) and low efficiency in the study of oligobacillary material, the presence in it of modified forms of the pathogen. In addition, the identification of isolates by conventional methods is expensive, laborious and requires sophisticated equipment. The use of the simplest method of identifying the isolated cultures in the agglutination reaction is limited to the types of non-tuberculous mycobacteria that give a stable suspension of the bacterial mass. The method according to patent No. 2193068 VI is not applicable to tuberculosis pathogens of mammals that form dry, difficult to disperse colonies.

The greatest reliability was demonstrated by a method for the diagnosis of tuberculosis, which is the prototype of the claimed invention (patent IA No. 43467 dated December 17, 2001).

The essence of this method is that the biological material is prepared for sowing and sown on a nutrient medium. The biological material is treated with a growth stimulator for 24-48 hours at a temperature of 36-38 ° C, after which, together with a growth stimulator, it is sown on nutrient medium, including enzymatic peptone, potato starch, agar-agar, sodium bicarbonate, and a calcium-dependent enzyme stimulator, and the nutrient medium is prepared just before seeding. The disadvantages of this method are the difficulties and errors of diagnosis, due to the difficulties of identifying the causative agent of tuberculosis, as by morphology, cultural, biochemical and pathogenic properties, isolates from the VCG environment are not only difficult to identify as the causative agent of tuberculosis, but also attributed to the genus Musclerassus. To verify the results, it is necessary to treat the culture with 10% caustic sodium, reseeding Levenshteyn-Jensen medium without malachite green with subsequent 1-2 months cultivation and identification of the grown culture in a bioassay, by biochemical tests or by the method of Poland (5).

The problem solved in the framework of the present invention is the creation of a method to simplify, reduce the cost, reduce the time of analysis and improve the accuracy of identifying the causative agent of tuberculosis.

The task is solved in the method of detecting tuberculosis infection by incubating the biomaterial in the growth stimulator of VCG, sowing it on the nutrient medium of VCG and cultivation, after which the grown culture is mixed with antiserum to the antigens of the mammalian tuberculosis pathogen and negative control serum to conduct an agglutination reaction and form a character agglutination and create a character agglutination and create a character agglutination and create a character agglutination and create a character agglutination. judge the culture of the causative agents of tuberculosis in mammals, and in the method of antiserum obtained by inje tions animals disintegrated without heating Mycobacterium tuberculosis with subsequent adsorption of its cultures atypical mycobacteria and banal microflora grown in a nutrient medium VCG, or it can be obtained by injecting an animal pathogen mammalian tuberculosis, grown on a nutrient medium VCG, followed by adsorption cultures atypical mycobacteria and banal microflora grown on a nutrient medium VKG.

In addition, the subject of this invention is a method for detecting tuberculosis infection by incubating the biomaterial in a growth stimulator of VCG, sowing it on the nutrient medium of VCG and cultivation, in which after cultivation the grown culture is mixed on slides with purified antibodies to antigens of the mammalian tuberculosis pathogen isolated from antisera , and negative control serum for the agglutination reaction and the formation of a characteristic agglutinate is judged on the identity of the cult It is the causative agent of tuberculosis in mammals, whereby the antiserum is obtained by injecting animals into disintegrated microbes of tuberculosis without heating, followed by adsorption of atypical mycobacteria and banal microflora grown on a nutrient medium with HCG, or antisera can be obtained by injecting animals with pathogenic organisms. VKH, followed by adsorption by its cultures of atypical mycobacteria and banal microflora grown on nutritionally th environment WKG.

Example

For the implementation of the method, the strain M. laf18 UA11 is grown on Soton medium for 6 weeks. For inactivation, phenol is added to crops to a concentration of 3-5%. After 48 hours, the suspension of bacterial mass in the culture fluid is treated with ultrasound for 15–20 min (15–35 kHz, up to 100 W / cm). Disintegrate in the ratio of 1: 3 is mixed with Freund's incomplete adjuvant and at a dose of 300-500 mcg / kg (protein

- 1 009817 per) is injected into sheep or young cattle at 2-3 points subcutaneously. Injections are repeated up to 11 times with an interval of 12-14 days. After 8-10 days after the last injection, blood and serum are obtained in animals in a conventional manner.

The resulting antiserum is tested for activity and specificity. As a negative control, healthy animal serum was used. For this culture of mycobacteria M. ou15 UA11ee, M. оуou15 8, BCG-1, M. 1bigi1155 Η ₃₇ ν, M. Auscht 1603, M. Gogiyit 342, 81ary, aigeik Co \\ 'an 1, E. soy is mixed in sterile vials with growth stimulator VCG at the rate of 1 mg per 1 ml of stimulator. The mixture is incubated for 48 hours at 37 ° C and plated on a VCG nutrient medium poured into Petri dishes. Plates are incubated at 37 ° C until colonies appear.

On a glass plate with a smooth, defatted surface, 20 μl of antisera and negative control serum are applied. To them add 1 bacteriological loop of bacterial mass of colonies of the indicated strains and mix thoroughly. Over the course of the reaction is monitored by viewing the plate with the help of the lower illumination of the illuminator OI-19 (Table 1).

Table 1

The specificity of antisera in RA

Types of mycobacteria, grown on medium VCG Negative serum Antiserum M.Lootz Μ.βονίί Уа11ее 0 ++++ BCG-1 0 ++++ Μ.Βονΐί 8 0 ++++ М.1иБегси1о815 Η ₃₇ Κν 0 ++++ 8 (arb. Aegeiz Soi / an 1, + ++++ E.soP ++ ++++ M. Gogishit 342 + +++ MLegge 17522ATSS 0 +++ M.Ausht 1603 0 +++ M. rYe! 1889 0 ++++ M.t1gas11i1age 5658 0 ++++

++++ - clear agglutination with the formation of coarse-or small-flocked agglutinate with clarification of the liquid and the formation of a characteristic pattern after drying and staining;

+ - the formation of small flakes without clarification of the liquid.

Due to the presence of agglutination with strains of non-tuberculous mycobacteria, as well as non-mycobacterial flora, antisera and negative serum are adsorbed by cultures giving agglutination. For this, the colonies of the respective cultures are removed in a nutrient medium with a BKH bacteriological loop and suspended for 24 hours in a 3% aqueous solution of phenol at the rate of 5-10 mg / ml. After inactivation, the cultures are precipitated by centrifugation at 3000 d for 10-15 min. The supernatant is removed. The bacterial mass of the culture is introduced into the antiserum at the rate of 2-10 mg / ml. The mixture is incubated for 1 hour at 37 ° C and 10-12 hours at 3-10 ° C. The bacterial mass is removed by centrifugation at 3,000 d for 10–15 min, the antiserum is checked by agglutination (Table 2).

table 2

Specificity of depleted sera in RA

Types of mycobacteria, grown on medium VCG Negative serum Antisera .Πονίδ Μ.6ονΪ8 УАПее 0 ++++ BCG-1 0 _ | - | - Μ.Βονΐδ 8 ++++ М.1иБегси1о515 Η ₃₇ Κν 0 ++++ 81ar. Agesiz Sochuap 1, 0 four E.soP 0 0 MToPiPit 342 0 . + MLegge 17522ATSS 0 0 M.Ausht 1603 0 0 Μ.ρΠΙεί 1889 0 0 M.1P1Gase11i1age 5658 0 +

++++ - clear agglutination with the formation of coarse-or small-flocked agglutinate with clarification of the liquid and the formation of a characteristic pattern after drying and staining;

+ - the formation of small flakes without clarification of the liquid.

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If necessary, the adsorption process is repeated. The finished serum is lyophilized or preserved by conventional methods.

To detect tuberculosis infection, sterile blood is mixed in equal volumes with a growth stimulator of VCG, and incubated for 48 hours at 37 ° C, and sown on freshly prepared VCG medium, poured into 15 ml in Petri dishes and 5.5 ml in short tubes with rubber stoppers . Crops are placed in a thermostat at 37 ° C.

In the presence of tuberculosis infection after 2-5 days on the surface of the medium VCG register the growth of rumination or waxy colonies. In smears stained with Ziehl-Nielsen, polymorphic sticks and cocci, branching and spherical structures of blue color are found.

To identify isolates, antisera and control negative serum are applied in 20 μl each on a glass plate with a defatted surface. The bacterial mass of the studied colony is mixed with antiserum and negative control serum. The reaction is taken into account in 5-7 minutes, using the lower illumination OI-19.

In tab. 3 shows the results of blood culture of 20 cows that reacted to tuberculin of a herds of tuberculosis unfavorable and a study of isolates in RA.

Table 3

Inv. cow number Tuberculin Reaction in mm Sowing on Wednesday VKG TB changes at slaughter Test tubes Petri dish RA 6119 five 0 + 4-4-4- 49/849 four four- P 4-4-4- 6541 3 + four- 4-4-4- 3224 four + four- + 44- + 3797 3 + 0 4-4-4-4- 3253 6 + P 4-4-4- 3207 ten + four- + 4- + 3436 five four- P 4 + 4- 947/147 3 + four- four- 4-4-4-4- 740 four four- 0 44- 3228 6 P P - 800 7 four- + four- 1000 eight + + four- 4-4-4-4- 3071 6 0 four- + 4-4- 400 3 four- four- ++ 7170 four four- 0 +++ 1200 four 0 0 - 984 five + four- ++++ 800 6 four- four- ++++ 7199 9 + + ++++ Control 0 0 0 0

- no growth; + - characteristic growth;

n - sprouts.

The results show that animals are infected with the causative agent of tuberculosis even in the absence of visible tuberculous changes during slaughter.

When sowing material in Petri dishes, the growth of the pathogen is obtained in 60%, the outside germination of microflora in 20% and a negative result in 20%. When the material is sown on test tubes, the growth of the pathogen is obtained in 85%, the outgrowth of extraneous microflora in 5% and a negative result in 10%.

To facilitate taking into account the results of the agglutination reaction, 10 μl of a K-250 solution of brilliant blue with a pH of 7 is added to the culture mixture with serums. In case of a negative reaction, the paint quickly drips apart, while the intensity of staining decreases. With a positive reaction, the intensity of staining does not change, and the agglutinate turns blue;

slides on which the reaction was set, dried in air, 1-2 s fixed over the flame, stained with carbolic fuchsin (1: 5) for 3-5 minutes, decolorized with 3% hydrochloric acid alcohol for 30-60 s, washed distilled water and dried. In the event of a positive reaction, a painted surface along the contour of the drop with characteristic discontinuities extending from the center remains on the slide. In case of a negative reaction, the color is absent, the contour of the drop is maintained or it is homogeneous without breaking (figure).

-3 009817

Thus, the conducted studies confirm the possibility of increasing the effectiveness of bacteriological diagnosis of tuberculosis, as evidenced by the correctness of the methods and technical solutions underlying the proposed method.

The method was successfully tested by the regional veterinary laboratories of the Republic of Belarus, in the bacteriological laboratory of anti-tuberculosis institutions in Kiev.

Claims (6)

CLAIM 1. A method for detecting tuberculosis infection by incubating biomaterial in a growth promoter of VKH, seeding on a culture medium of VKH and culturing, characterized in that after cultivation, the grown culture is mixed with antiserum to antigens of the pathogen of mammalian tuberculosis and a negative control serum for the agglutination reaction and to form a characteristic agglutinate is judged on the belonging of the culture to the pathogens of mammalian tuberculosis. 2. The method according to claim 1, characterized in that the antiserum is obtained by injecting mycobacterium tuberculosis disintegrated without heating into animals, followed by adsorption of atypical mycobacterium cultures and commonplace microflora grown on VKG nutrient medium. 3. The method according to claim 1, characterized in that the antiserum is obtained by injecting an animal with the causative agent of mammalian tuberculosis grown on VKG nutrient medium, followed by its adsorption of atypical mycobacteria and common microflora cultures grown on VKG nutrient medium. 4. A method for detecting tuberculosis infection by incubating biomaterial in a growth promoter of VKH, plating on a culture medium of VKG and culturing, characterized in that after cultivation, the grown culture is mixed on slides with purified antibodies to mammalian tuberculosis pathogen antigens isolated from antiserum and a negative control serum for the agglutination reaction, and the formation of a characteristic agglutinate judge the culture belongs to the causative agents of tuberculosis them. 5. The method according to claim 4, characterized in that the antiserum is obtained by injecting mycobacterium tuberculosis disintegrated without heating into animals, followed by adsorption of atypical mycobacteria and banal microflora grown on a VKG nutrient medium. 6. The method according to claim 4, characterized in that the antiserum is obtained by injecting an animal with the causative agent of mammalian tuberculosis grown on VKG nutrient medium, followed by its adsorption of atypical mycobacteria and common microflora cultures grown on VKG nutrient medium.