Complex allergen for differentiating dried purified tuberculine allergic reactions for mammals

FIELD: medicine, veterinary science.

SUBSTANCE: invention concerns veterinary medicine. Currently, for differentiating nonspecific tuberculin reactions in cattle, dried purified tuberculin is used for mammals and "КАМ" with considering the sensitisation pattern by the reaction intensity. A common complex allergen is produced by protein settling from M scrofulaceum No. 12-C and M intracellulare No. 13-H strain cultures with added allergen produced from Corynebacterium xerosis N1911, in amount 1350 units of activity. The presence of coryneformic bacteria allergen in the "КАМ" composition improves the efficacy of a simultaneous dried purified tuberculin test for mammals in differentiating the nonspecific coryneformic bacteria reactions.

EFFECT: use of the declared allergen allows to prevent unreasonable slaughter, as well as further diagnostic finding expenses.

2 tbl

 

The invention relates to the field of veterinary medicine and recommended to use for differentiation of allergic reactions to PPD-tuberculin for mammals.

In practice, allergic tests for tuberculosis numerous cases are known when responding to PPD - tuberculin cattle, pathological and bacteriological studies to detect tuberculosis and cannot cause sensitization remains outstanding.

It is established that the main cause of the manifestations of reactions to tuberculin in healthy animals is cause sensitization by atypical mycobacteria, have common antigens from Mycobacterium tuberculosis[1, 2, 3].

In addition, the cause sensitization of the microorganism to tuberculin may be of Nocardia and rhodococci with common radiopacities data with mycobacteria [5, 6].

Great interest in this regard are Corynebacterium, with mycobacteria General physico-chemical and biological properties and widespread in nature [4, 10]. There are reports [6, 11] that is infected with Corynebacterium animals react to PPD-tuberculin for mammals.

The variety of reasons that contribute to the sensitization of animals to tuberculin complicates the differential diagnosis of tuberculosis that eventually the equipment is foreseen significant economic losses for farmers.

The mentioned phenomenon necessitates the determination of the specificity of allergies, the results of which can lead to increased efficiency allergic method in the diagnosis of tuberculosis in animals.

For allergic diagnosis of tuberculosis in cattle using PPD - tuberculin for mammals, for which M. bovis, strain No. 8, is grown on the environment Sotona, within 2 months [8]. Differentiation sensitization caused by avian species Mycobacterium carried out in the simultaneous assay, using purified tuberculin for birds and PPD-tuberculin for mammals. Tuberculin for birds made from M.avium (strain No. 2282), and the difference in the intensity of reactions determine the cause sensitization.

Known allergens derived from Nocardia and Rhodococcus [5]that the complex allergen improve the efficiency of simultaneous samples with PPD-tuberculin during differentiation of nonspecific reactions. In addition, the allergen from N.asteroides used for diagnosis of allergic nocardiosis.

The technical nature closest to the claimed allergen is a comprehensive allergen from atypical mycobacteria (KAM), consisting of M.scrofulaceum No. 12-C and M.intracellulare No. 13-H [8]. To obtain allergen culture grown on synthetic medium Sotona, precipitated protein with trichloroacetic acid and periostat ammonium sulfate. In the future, to remove salts dialysis through a cellophane membrane against demineralized water.

The content of units (ED.) in the solution of protein of each species is determined on an infected gomologicnami mycobacteria Guinea pigs in comparison with drugs known activity. Thus obtained nonallergenic mixed in equal quantities in content units. Despite the expressed differentiating properties compared with tuberculin for birds, a comprehensive allergen from atypical mycobacteria has a significant disadvantage: the efficiency depends on the degree of relatedness of the organisms that caused the sensitization and mycobacteria, used for the manufacture of allergen[9].

The aim of the present invention is to increase the efficiency of differentiation of allergic reactions in simultaneous sample with the CAM by expanding its antigenic structure.

This goal is achieved by the fact that part of the complex allergen from atypical mycobacteria containing antigens from M.scrofulaceum No. 12-C and M.intracellulare No. 13-H, entered the antigen from Corynebacterium (Corinebacterium xerosis N1911).

Comparative analysis of the composition of allergens leads to the conclusion that the claimed allergen differs significantly from the known content more allergens by introducing corynebacteriaceae allergen. T is thus, declare the allergen is significantly different from the prototype and therefore meets the criterion of "novelty."

For the manufacture of allergen culture of Corynebacterium (Corynebacterium xerosis N1911) were grown on synthetic medium Sotona with a mixture of individual n-alkanes, content in the chain from 10 to 17 carbon atoms, for 2 months. It should be noted that the environment Sotona us was modified and comparatively tested previously. The results of these tests showed a higher efficiency of the modified variant for the cultivation of Corynebacterium, the biomass of which exceeded the control series more than 2 times. It is possible to obtain 2 times more active protein per unit of volume. Flasks with culture, where the thickness of the layer of Bakassi reached about 1 cm, autoclaved at 1.5 ATM for 30 minutes Separated the bacterial mass by filtration and centrifugation, and then spent the precipitation of the protein. The volume of supernatant in the amount of 1.5 liters deposition in the isoelectric point of NaCl (18% concentration, at PH 4,1) received a 3.2 grams of protein. The precipitate was washed, dried, Packed up in glass vials and kept in the refrigerator.

Subjects concentration of protein(0,00005; 0,0001; 0,0002; 0,0003; 0,0004 and 0.0005 mg in 0.1 ml) was received, diluting 0.01 ml (0.001 g) of a solution of 10% concentration in sterile saline (1: 1000). After mixing, 0.1 ml of races the thief made alternately in tubes with saline solution (9,9; 4,9; 2,9; 2,4; 1,9 and 19.9 ml), having thus, breeding, 1:100, 1:50, 1:30, 1:25, 1:20, and 1:200 respectively.

The definition of the threshold sensitivity of the allergen was performed on 24 Guinea pigs, 4 were in the control, 25 days after subcutaneous infection with Corynebacterium, in the dose of 10 mg wet culture in 1 ml of physiological solution. Each titrated dose of allergen, 0.1 ml was injected intradermally on depilitory plot of the lateral costal surface. The reaction was evaluated at 32 hours after injection. This was determined by the intensity of the reactions (in mm2) in diameter papules, to calculate the area-averaged value of the intensity of reactions to a certain protein concentration. The results obtained are presented in table 1.

Guinea pigs of the control group for allergens did not respond. The protein concentration 0.00005 mg reaction was one Guinea pig intensity 1.8 mm2. In the second group at a concentration of 0.0001 mg reacted with 2 Guinea pigs with an average intensity of 4.6 mm

Otherwise, the protein concentration is 0.0002; 0,0003; of 0.0004 and 0.0005 mg reacted all experimental animals, and has not established a direct relation reactions from further increase protein allergen.

Thus, the threshold sensitivity of the allergen is within 0.00005 mg in 0.1 ml of solution, which rises up to the of centrali 0.0003 mg and in the future regardless of the increase, the intensity of the reaction is reduced. Therefore, per unit protein of Corynebacterium was adopted dose of 0.0003 mg in 0.1 ml.

Declare the allergen was prepared on the basis of protein content in CHAM-e - 1350 units steps in 0.2 ml and 0.0003 mg protein corynebacteria in 0.1 ml, which has been accepted by us per unit. This took 20.25 mg wet culture Corynebacterium xerosis and mixed with 10 ml CAM. Thus, received 10 ml of allergen consisting of proteins atypical mycobacteria and corynebacteria. To obtain a working solution, a content of 15 units of 0.1 ml, Guinea pigs, 0.2 ml (1350 u), dissolved 8.8 ml of saline (1:45).

The test derived allergen was performed on infected myobacterial (Scrofulaceum and BCG) and Corynebacterium (xerosis) Guinea pigs in 27 days after infection. Each culture were infected for 3 Guinea pigs and 3 were in control.

The study was performed in politesse. Experienced cavies on depilitory plot rib surface was administered to the test allergen on the one hand and CAM with another dose of 0.1 ml (10 IU). Evaluation of reactions was performed 24 hours after injection. The results are shown in table 2.

The greatest intensity of reactions in the target allergen was detected in Guinea pigs infected with Corynebacterium (117,17 mm2). The reaction on the CAM in this group was significantly is anise.

In other groups of Guinea pigs to study the allergen reacted less intensively, although KAM reaction was more pronounced.

Thus, infected with Corynebacterium Guinea pigs, the sensitivity of a comprehensive allergen content corynebacteriaceae allergen exceeds KAM 2 times.

Production testing of allergen spent in the economy, the environment where cows and heifers of different ages constantly detected reacting to tuberculin animals, but the results of simultaneous samples with KAM remains uncertain.

In simultaneous sample with the test allergen examined 14 respond positively to tuberculin animals from 64 presledovannych. When taking into account the results of the number of positively reacting (intense reaction to PPD-tuberculin) was 1 animal, reacting negatively (reaction to the tested allergen harder) - 12 goals. The number of reacting equally amounted to 1 pet. It follows that the difference in the intensity of reactions to tuberculin and to the tested allergen reliably and results of simultaneous samples are defined. Therefore, it is safe to say that the farm animals that have for a long time the results of simultaneous samples with KAM remained uncertain, sensitized respecof the český.

The obtained results allow to conclude that the sensitivity of the proposed allergen exceeds the sensitivity of the prototype (KAM) as in identifying infected with Corynebacterium animals and sensitized atypical myobacterial, while increasing the efficiency of simultaneous samples during differentiation of nonspecific reactions to PPD-tuberculin.

Thus, the use of the proposed allergen in simultaneous sample with CAM, allows you to prevent unnecessary slaughter of animals, to reduce the cost of conducting organizational-economic and veterinary-sanitary measures.

Sources of information

1. Cassic UA Study sensitizing and pathogenic properties of atypical mycobacteria // veterinary - 1989. No. 4. - S-15.

2. Marsha O., Tahns K.K. Parallelizes reactions to tuberculin and their differentiation // veterinary medicine. - 1978. No. 4. P.35-38.

3. March O.V., Ovdienko N.P., Tkachev-Kuzmin A.V. // Tuberculosis in farm animals. - M.: Agropromizdat, 1991. - P.28-32.

4. Nesterenko O.A. Nicardipine and carinatae bacteria. // Kiev: Naukova Dumka. 1985. 333 pages

5 Nurudinov R.A. Way of differentiating the diagnosis of tuberculosis. Patent for invention No. 2146946. - 1998

Moratinos R.A. Tuberculosis in cattle in the North Caucasus republics and Kalmykia (epizootology the Oia, diagnosis and control measures) Diss. Doc. wet. Sciences. Moscow - 1998. - 370 pages

7. Nurudinov R.A., Baratov MO, verdieu E.A. Comprehensive allergen for differentiation of allergic reactions in cattle on PPD - tuberculin for mammals. Patent for invention No. 2217165. - 2003

8. Balls A.N. and other Drugs for the diagnosis of tuberculosis in animals. // Kursk bio 100 formed. - 1996. - S-403.

9. Balls A.N. Allergic diagnosis of tuberculosis in animals: Increasing its effectiveness. Diss. Doc. wet. Sciences. Moscow - 1989

10. Ridll M-Serologycal relationships of Nocordia, Mycobacterium, Corynebacterium and the Rhodochrous taxon with special reference to taxonomy Goteborg. /1977. - 52p.

11. Shukia R., Nafh N., Singh G. Observations on non-specific reactions to tuberculin in sheep and goats with Coiynebacterium ovis "Experientia" 1971.27. No. 2. - 204-205.

Table 1
The dependence of the intensity of reactions to allergens from the concentration of protein in solution
The protein concentration in serum (mg / 0.1 ml)(Corynebacterium xerosis N1911),
The intensity of reactions, mm2M±M
-
0,00005 1,80,45±0,03
-
-
6,3
0,0001-4,6±0,8
12,4
-
21,6
is 0.000230,428,4±8,6
28,2
the 33.4
90,3
0,000372,483,2±18,9
87,2
83,1
86,3
0,0004of 60.5to 70.7±15,3
64,8
71,2
70,7
0,000550,360,4±13,2
64,2
58,4

Table 2
The intensity of the reactions in infected Guinea pigs to study the allergen
No.View infected cultureNumber of animals in the experienceThe intensity of the reactions in mm2on
Comprehensive allergenM±M KAMM±M
1126,12±18,967,34±7,6
1Corynebacterium xerosis2119,18±16,475,62±8,365,05
117,17
3106,22±12,852,19±6,7
197,65±11.299,70±11,9
2Mycobacterium scrofulaceum2100,16±12.195,5596,54±10,395,64
391,84±9,790,69±9,6
1108,76±13,1100,64±12,6
3Mycobacterium BCG293,53±10,270,6891.36±9,894,90
390,75±9,692,44±9,92
1--
4 Control2----
3--

Comprehensive allergen for differentiation of allergic reactions in cattle on PPD-tuberculin for mammals obtained by precipitation of the protein from the culture strain M.scrofulaceum No. 12-C and M.intracellulare No. 13-H, characterized in that it further contains the allergen from Corynebacterium obtained from the culture of Corynebacterium xerosis strain No. 1911, in the amount of 1350 units of action.



 

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