RU2443428C1 - Method for making allergen for differential diagnostics of para-allergic responses in cattles on ppd tuberculin for mammals - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to veterinary microbiology. A method for growing atypical mycobacteria cultures on a liquid nutrient medium, culture inactivation by autoclaving at 120°C for 30 minutes, protein settling from a culture filtrate of each strain separately by trichloroacetic acid to the final concentration of 4%, re-settling by ammonium sulphate at 50% saturation, protein dialysis through a cellophane coating as against deionised water to remove salts; analysis of the protein solution for its activity in each atypical mycobacteria culture, mixing of the protein solutions in equal portions by the "ED" concentration, sterilising filtration of the solution, packaging and lyophilisation. As atypical mycobacteria cultures, M. intracellulare, M. scrofulaceum, M. fortuitum and M. smegmatis are used, while the atypical mycobacteria cultures are grown on the liquid nutrient medium for 2-3 weeks.

EFFECT: method provides high-quality differential diagnostic response on PPD tuberculin for mammals and cutting economic loss of induced PPD tuberculin responded animal killing.

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Description

The present invention relates to the field of veterinary microbiology, in particular to a method for producing an allergen from atypical mycobacteria for the diagnosis of tuberculosis in a simultaneous test with PPD tuberculin for mammals.

The main and generally accepted method for the diagnosis of cattle tuberculosis is an intradermal test using tuberculin PPD. One of the most important problems with this diagnosis is the appearance of non-specific reactions to tuberculin. Massive detection of non-specific reactions to tuberculin leads to the slaughter of a large number of healthy animals, which increases the extent of economic damage and raises doubts about the correct diagnosis of tuberculosis in a known manner.

In connection with the widespread non-specific (para-allergic) reactions to tuberculin in different years, many authors have proposed various methods for differentiating these reactions.

Numerous studies by domestic and foreign authors have established that the main reason for the manifestation of nonspecific reactions in cattle to tuberculin is sensitization by mycobacteria: M. avium, M. paratuberculosis and rapidly growing atypical mycobacteria, widely distributed in the environment (3, 4, 5).

Since 1978, in our country, to simulate differentiation of nonspecific reactions, a simultaneous test with the use of TPD tuberculin for mammals and a complex allergen from atypical mycobacteria (KAM) has been used, and since 2002, a simultaneous test with PPD tuberculin for mammals and PPD tuberculin for birds has been used.

A known method of manufacturing an allergen for differentiating allergic reactions to PPD tuberculin for mammals, comprising mixing nocardium protein 0.54 mg and rhodococcal protein 0.405 mg in an ampoule with 10 ml of KAM. At the same time, 0.2 ml of the obtained allergen contained 1350 units of action of each of the constituent components. To prepare a working solution (10 units of action for guinea pig), 1 ml of the drug was transferred into a test tube with 9 ml of solvent (physiological solution). After stirring, 2 ml from the first tube was transferred to the second with 11.5 ml of solvent. Thus, in the second test tube, a solution containing 0.1 ml of 10 PIECES of each component (1) was obtained.

There is also a method of manufacturing a complex allergen from atypical mycobacteria (KAM), including the cultivation of mycobacteria M. scrofulaceum No. 12-C and M. intracellulare No. 13-N (3) on a liquid nutrient medium at 37-38 ° C for 8 weeks, inactivation cultures by autoclaving at a temperature of 120 ° C for 30 minutes, precipitating the protein from the culture filtrate with trichloroacetic acid and reprecipitating it with ammonium sulfate, followed by dialysis through a cellophane membrane to remove sulfates. The content of units of action (ED) in 1 ml of a protein solution of each type of these mycobacteria is determined on guinea pigs; sensitized by homologous mycobacteria in comparison with the reference series of these drugs with known activity. The obtained monoallergens are mixed in equal amounts according to the content of action units (2). Prototype.

Complex allergen (CAM), which includes atypical mycobacteria of only the 2nd and 3rd groups according to the classification of the Runion, does not provide the completeness of the differential diagnosis.

An analysis of bacteriological studies conducted in the VIEV showed that mycobacteria isolated from pathological material from tuberculin reacting to PPD, as well as from animals killed for diagnostic purposes from tuberculosis-free farms, in most cases are fast-growing atypical mycobacteria of the 4th group of classification according to Runon.

Reporting data from production veterinary laboratories confirm these results. Atypical mycobacteria are isolated in almost the entire territory of Russia in veterinary laboratories, which are distributed as a percentage as follows: 1st group - 2%; 2nd - 19.7%; 3rd - 16.8% and 4th group - 59.5%, that is, the most frequently allocated 4th group according to the Runon (up to 60%) of atypical mycobacteria (M. fortuitum, M. smegmatis ), which cause sensitization to tuberculin in guinea pigs, cattle, sheep, goats and rabbits (3, 4, 5, 6, 7).

The research objective was to increase the effectiveness of a simultaneous test with PPD tuberculin for mammals and CAM, as well as reducing the time required to grow atypical cultures.

This was achieved due to the fact that in the manufacture of a complex allergen from atypical mycobacteria (KAM), 2 types of Mycobacteria of the 4th Ranon group (M. fortuitum, M. smegmatis) were additionally added, while the cultures were grown on Soton’s liquid nutrient medium during 2-3 weeks, not 8 weeks.

A method of obtaining the proposed allergen is as follows: the cultivation of atypical mycobacteria cultures M. scrofulaceum, M. intracellulare, M. fortuitum and M. smegmatis are carried out separately on Soton’s liquid nutrient medium at a temperature of 37-38 ° C for 2-3 weeks, inactivate the culture autoclaving at 120 ° C for 30 minutes, precipitate the protein from the culture filtrate of each strain individually with trichloroacetic acid to a final 4% concentration, reprecipitate the protein with ammonium sulfate at 50% saturation followed by dialysis of the black protein solution without a cellophane membrane against demineralized water to remove sulfates, determination of protein concentration in solutions of each type of mycobacteria with subsequent titration of the concentration on guinea pigs sensitized by various types of mycobacteria within 0.45-0.5 mg in 0.1 ml, which corresponds to an activity of 1350 units action (UNIT), then mix protein solutions in equal proportions according to the UNIT content, sterilely filter, pack and lyophilize.

Specific examples are shown in tables 1, 2 and 3.

Table 1 Test results of various monoallergens on atypical mycobacteria sensitized guinea pigs (n = 25) Allergen
The amount of protein in 0.1 ml tested. raster The intensity of the reactions of animals to the homologue, monoallergens (cf. ⌀ mm) Sensitization M.intracellulare Sensitization.
M. scrofulaceum Sensitization. M.fortuitum Sensitization of M. smegmatis Allergen intracellulare 19.4 ± 1.37 6.5 ± 1.05 5.1 ± 1.05 4.7 ± 0.63 Allergen scrofulaceum 6.3 ± 0.54 20.3 ± 2.15 5.8 ± 1.14 7.3 ± 1.4 Allergen fortuitum 7.6 ± 1.23 5.2 ± 0.96 29.7 ± 2.68 11.7 ± 2.37 Allergen smegmatis 6.3 ± 0.67 5.8 ± 0.96 12.4 ± 2.3 31.4 ± 3.87 PPD tuberculin for mammals. 4.1 ± 0.85 3.2 ± 0.54 5.8 ± 1.06 6.2 ± 1.52

The data in Table 1 show that allergic reactions to monoallergens (MA) from the tested allergen in guinea pigs sensitized by homologous cultures are greater than for MA included in the production of KAM:

MA from M.fortuitum — 53.1% more than MA from M. intracellulare, and 46.3% more than MA from M. scrofulaceum;

MA from M.smegmatis increased by 62.4 and 54.7%, respectively.

table 2 Test results of various allergens on atypical mycobacteria sensitized guinea pigs (n = 25) Allergen The intensity of the reactions of animals to the homologue, monoallergens (⌀ mm) Sensitization M.intracellulare Sensitization. M.scrofulaceum Sensitization. M.fortuitum Sensitization M.smegmatis PPD tuberculin for mammals. 4.1 ± 0.85 3.2 ± 0.54 5.8 ± 1.06 6.2 ± 1.52 Production KAM 17.6 ± 3.54 15.6 ± 3.85 7.2 ± 4.83 6.3 ± 0.55 Test allergen 18.2 ± 2.14 18.9 ± 1.76 20.8 ± 2.88 21.5 ± 2.75

In comparative studies of the test drug and production KAM in animals sensitized with M.fortuitum and M.smegmatis, an increase of 3 times was observed in allergic reactions. In animals sensitized with M.intracellulare and M.scrofulaceum, reactions to the test allergen were 3.4 and 21.2% higher, respectively.

Table 3 Comparative tests of an allergen obtained from 4 types of atypical mycobacteria and production of KAM in a simultaneous test for PPD tuberculin for mammals in cattle Allergen Animals that respond to PPD tuberculin for mammals (n = 26) Number of animals (animals) that respond to the allergen Percentage of reacting animals Intensity of reactions, skinfold thickness (mm) Test allergen 22 84.6 13.1 ± 1.14 Production KAM fourteen 53.8 8.3 ± 1.25 TPD tuberculin for mammals four 11.5 3.2 ± 0.45

In a planned study of cattle (457 animals), a successful tuberculosis farm found that 26 cows reacted to TPD for mammals.

After 30 days, when re-examining these 26 cows in a simultaneous test with PPD tuberculin for mammals, production KAM and the test allergen, it was found that out of 26 animals, 14 responded to the production KAM with an increase in the thickness of the skin fold to 8.3 mm, to the test allergen - 22 s an increase in the thickness of the skin fold to 13.1 mm. At the same time, a reaction was observed in 3 animals with TPD tuberculin for mammals with an increase in the thickness of the skin fold from 2.8 to 3.0 mm. From this it can be concluded that the reactions in cattle to TPD tuberculin for mammals were para-allergic, that is, the animals were sensitized with atypical mycobacteria.

The proposed method was tested by us with a positive result in laboratory conditions on 50 guinea pigs sensitized with various mycobacteria and production conditions on cattle (457 animals) of a tuberculosis-free farm that responds to TPD tuberculin for mammals.

The proposed method for producing a complex allergen will reduce the time of growing crops from 8 to 2 weeks, will be widely used in livestock farms of the Russian Federation, allowing to improve the quality of differential diagnostics for tuberculin PPD for mammals and reduce the economic damage from forced slaughter of tuberculin animals that respond to PPD.

Information sources

1. Patent for invention No. 2217165, of the Russian Federation, А61К 39/352002. A complex allergen for the differentiation of allergic reactions in cattle on PPD tuberculin for mammals / Nuratinov R.A. et al., 2002.

2. Dis ... doc. vet. sciences. Allergic diagnosis of tuberculosis in animals: increasing their effectiveness / Sharov A.N., Moscow. - 1989. - P.75. (Prototype).

3. G. Veterinary Medicine. - 1978 - No. 4. - S. 35-38.

4. Collection. scientific Proceedings of VGNKI. - M., 1985, S.3-9.

5. Dis ... doc. vet. sciences. Cattle tuberculosis in the republics of the North Caucasus and Kalmykia (epizootology, dianostics and control measures) / Nuratimov R.A., Moscow. - 1998. - P.370.

Claims (1)

A method of manufacturing an allergen for the differential diagnosis of para-allergic reactions in cattle on PPD tuberculin for mammals, including growing atypical mycobacteria cultures in a liquid nutrient medium, inactivating cultures by autoclaving at 120 ° C for 30 minutes, precipitating protein from the culture filtrate of each strain individually trichloroacetic acid to a final 4% concentration, reprecipitation with ammonium sulfate at 50% saturation, dialysis of a protein solution through a cellophane coating a sample against demineralized water to remove salts, determining the activity of the protein solution of each culture of atypical mycobacteria, mixing protein solutions in equal proportions of the ED content, sterilizing the filtration of the solution, packaging and lyophilization, characterized in that M. intracellulare, M is used as the culture of atypical mycobacteria scrofulaceum, M. fortuitum and M. smegmatis, and the cultivation of atypical mycobacteria cultures in a liquid nutrient medium is carried out for 2-3 weeks.