RU2538690C1 - Method of manufacturing allergen for differential diagnostics of para-allergic reactions in cattle to ppd tuberculin for mammals - Google Patents

Abstract

FIELD: veterinary medicine.

SUBSTANCE: method of manufacturing allergen for differential diagnostics of para-allergic reactions to PPD tuberculin for mammals consists in the fact that for the separate deposition of protein, trichloroacetic acid to a final concentration of 6-8 % is taken from the culture filtrate, the protein solution is left for 12-20 hours at a temperature of 4-6°C, centrifuged at 4-8 thousand rev/min for 30 minutes, dissolved in distilled water with pH 7.0-8.0 and the resulting protein solution is put on dialysis through a cellophane wrapper against demineralised water for 12-24 hours, after which 0.2-0.3% phenol is added for stabilisation, the preparation is sterilised and packaged in its native state.

EFFECT: reduction of time of manufacturing the complex allergen from atypical mycobacteria, improving the quality of differential diagnostics of PPD tuberculin for mammals, and enables to reduce the economic damage from forced slaughter of animals reacting to PPD tuberculin.

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Description

The present invention relates to the field of veterinary microbiology, in particular to a method for the manufacture of an allergen for the diagnosis of para-allergic reactions to tuberculosis in a simultaneous test with PPD tuberculin for mammals.

A common method for diagnosing 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 ways to differentiate these reactions.

Numerous studies by domestic and foreign authors have established that the main reason for the manifestation of non-specific reactions in cattle to tuberculin is sensitization by mycobacteria: M. avium, M. paratuberculosis and rapidly growing atypical mycobacteria, which are widespread in the environment (1, 2, 4).

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 a complex allergen from atypical mycobacteria (KAM), including growing mycobacteria M. scrofulaceum No. 12-C and M. intracellulare No. 13-H in a liquid nutrient medium at 37-38 ° C, inactivation of cultures by autoclaving at a temperature of 120 ° C for 30 minutes, precipitation of the protein from the culture filtrate with trichloroacetic acid and reprecipitation with ammonium sulfate, followed by dialysis through a cellophane membrane to remove sulfates. The content of action units (UNITS) in 1 ml of a protein solution of each species of these mycobacteria was determined on guinea pigs sensitized by homologous mycobacteria in comparison with the reference series of these drugs with known activity. The obtained monoallergens were mixed in equal amounts according to the content of action units (1).

There is also known a method for producing an allergen, which consists in the following: growing cultures of atypical mycobacteria M. scrofulaceum, M. intracellulare, M. fortuitum and M. smegmatis were carried out separately on Soton’s liquid nutrient medium at a temperature of 37-38 ° C for 2-3 weeks, the cultures were then inactivated by autoclaving at 120 ° C for 30 minutes, the protein was precipitated from the culture filtrate of each strain individually with trichloroacetic acid to a final 4% concentration, the protein was reprecipitated with ammonium sulfate at 50% saturation, followed by dialysis protein through a cellophane membrane against demineralized water to remove sulfates, then the protein concentration in the solutions of each strain and activity were determined by the content of action units (ED) on guinea pigs sensitized by various types of mycobacteria and protein solutions were mixed in equal proportions by the content of ED, sterilely filtered, packaged and lyophilized (Prototype).

The objective of our research was to simplify the method, reduce the time spent on it and getting the drug in its native state.

This can be achieved due to the fact that in the manufacture of a complex allergen (KAM) for separate precipitation of proteins from the culture filtrate, trichloacetic acid is taken to a final concentration of 6-8%, a protein solution is left for 12-20 hours at a temperature of + 4-6 ° C, centrifuged at 4-6 thousand rpm. within 30 minutes, dissolved in distilled water with a pH of 7.0-8.0 and the resulting protein solution put on dialysis through a cellophane film against demineralized water for 12-24 hours, after which 0.2-0.3% phenol is added to stabilization, filtered sterilely and packaged in the native state.

The proposed method for producing an allergen is as follows: growing cultures of atypical mycobacteria M. scrofulaceum, M. intracellular, M. fortuitum and M. smegmatis are carried out separately on Soton’s liquid nutrient medium at a temperature of 37-38 ° C for 4-6 weeks, inactivate the culture by autoclaving at 120 ° C for 40-60 minutes, the protein is precipitated from the culture filtrate separately with trichloroacetic acid to a final concentration of 6-8%, left for 12-20 hours at a temperature of + 4-6 ° C, centrifuged at 4-6 thousand .rpm for 30 min, the resulting protein dissolving t in distilled water with a pH of 7.0-8.0, poured into cellophane tubes and put on dialysis against demineralized water to remove salts and acid residues for 12-24 hours, sterilized filtration is carried out, the protein concentration, the content of units of action (U ) in 1 ml of a protein solution obtained from each strain of mycobacteria with subsequent titration of the concentration on guinea pigs sensitized by homologous species of mycobacteria in comparison with the reference series of these preparations with known activity, then mix the solutions Tree equally in content units, is added 0.2-0.3% phenol for stabilization, sterile filtered and dispensed into the native state.

The proposed method for producing allergen allows to reduce the duration of the preparation of the drug by eliminating the reprecipitation of proteins by ammonium sulfate (2 days), reduce the duration of dialysis to one day, and to exclude the lyophilization of the drug, since the drug is packaged in its native state.

The allergen obtained by the proposed method was tested by us with positive results in laboratory conditions on sensitized guinea pigs (M. avium and BCG) with various atypical mycobacteria, 65 pigs and on cattle of the TB-free farms in the Volgograd, Yaroslavl, Ryazan regions (675 goals .).

The following are specific examples of the implementation of the proposed method.

Table 1 Test results of various monoallergens in atypical mycobacteria sensitized guinea pigs Allergen, the amount of protein in 0.1 ml test. raster The intensity of animal responses to the homolog. monoallergens (mm) Sensitization
M. intracellulare Sensitization.
M. scrofulaceum Sensitization
M. fortuitum Sensitization
M. smegmatis Allergen Intracellulare 14.3 ± 1.22 4.8 ± 0.42 8.5 ± 0.95 8.1 ± 0.94 Allergen Scrofulaceum 7.1 ± 0.65 19.3 ± 1.45 6.3 ± 0.34 7.4 ± 0.96 Allergen Fortuitum 7.6 ± 1.23 5.2 ± 0.96 15.7 ± 1.53 9.5 ± 1.65 Allergen Smegmatis 6.3 ± 0.67 5.8 ± 0.96 12.4 ± 2.3 21.2 ± 2.14 KAM productions. 3.4 ± 0.95 3.2 ± 0.66 5.8 ± 0.96 6.2 ± 0.97 Prototype 6.8 ± 0.97 13 ± 0.88 7.5 ± 0.96 14.3 ± 1.05

table 2 The test results of various allergens (control, prototype and claimed) on sensitized atypical mycobacteria guinea pigs Allergen The intensity of animal responses to the homolog. monoallergens (mm) Sensitization
M. intracellulare Sensitization.
M. scrofulaceum Sensitization
M. fortuitum Sensitization
M. smegmatis PPD tuberculin for mammals. 4.1 ± 0.76 3.8 ± 0.33 2.5 ± 0.46 4.1 ± 0.43 Production KAM 15.3 ± 1.34 14.4 ± 0.88 8.4 ± 1.03 9.6 ± 1.97 Prototype 17.0 ± 2.05 15.1 ± 1.74 16.4 ± 2.05 16.2 ± 1.86 Test allergen 16.7 ± 1.22 15.8 ± 2.05 17.2 ± 1.55 16.8 ± 2.35

Examples of tests in production conditions on cattle.

Example 1. We examined 256 cows of a prosperous tuberculosis farm (SPK named after Kirov, Staropoltavsky district, Volgograd region) with an intradermal breakdown at the same time as TPD tuberculin for mammals, KAM, prototype and the claimed allergen. The drugs were administered at a dose of 0.2 ml each into the neck (on the right side of the PPD and KAM, and on the left - the last 2). Accounting and evaluation of reactions were done 72 hours after drug administration. In 16 goals, reactions were more pronounced on the PPD tuberculin, on KAM - 29, and on the left side of the neck on both drugs reactions were pronounced in 33 goals. The total number of reacting animals (16 + 33) is 49 (indicator A). According to the table for determining the significance of differences, the figure 49 was found, which is 34 (indicator B), which indicates the significance of differences in reactions (94.5%), i.e. animals of these groups are sensitized by atypical mycobacteria, which was subsequently confirmed by bacteriological studies.

Similar tests of drugs in the same sequence were carried out on 250 heads of cattle (OAO PZ named after Dzerzhinsky, Yaroslavl region, Yaroslavl region) and 169 cows (collective farm Zaveta Ilyicha, Kasimov district, Ryazan region) and everywhere sensitization of animals by atypical mycobacteria is confirmed. The results of a simultaneous test were confirmed by bacteriological studies, i.e. the presence of tuberculosis in animals of the above farms is excluded.

An analysis of the data obtained during a comparative test of the claimed allergen, prototype and production KAM showed that the claimed allergen has greater sensitivity than the production KAM, and the same sensitivity as the prototype when tested on sensitized guinea pigs, and in production conditions on cattle.

The proposed method for producing allergen will find application in the biological industry and livestock farms, making it possible to shorten the production of a complex allergen from atypical mycobacteria (KAM), 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. Dis ... doc. vet. sciences. Allergic diagnosis of tuberculosis in animals: increasing their effectiveness / Sharov A.N., Moscow. - 1989. - P.75. (Analog).

2. Patent for invention No. 2443428, Russian Federation, A61K 39/04 (2012) and A61K 39/35 (2012). A method of manufacturing an allergen for the differential diagnosis of para-allergic reactions in cattle on PPD tuberculin for mammals (Gulukin M.I. et al.)

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

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

Claims (1)

A method of manufacturing an allergen for the differential diagnosis of paraallergic reactions to PPD tuberculin for mammals, characterized in that trichloroacetic acid is taken to a final concentration of 6-8% to separate the protein from the culture filtrate, a protein solution is left for 12-20 hours at a temperature of 4-6 ° C, centrifuged at 4-8 thousand rpm for 30 minutes, dissolved in distilled water with a pH of 7.0-8.0 and the resulting protein solution is put on dialysis through a cellophane membrane against demineralized water for 12-24 hours, after why add 0.2-0.3% phenol to stabilize, sterilize and pack the drug in its native state.