RU2383023C1 - Method of determining sensitivity of horses to antigens of microorganisms of various taxonomic keys - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method of determining sensitivity of horses to antigens of microorganisms of various taxonomic keys, which includes carrying out reaction of leukocytolysis with blood leucocytes using antigen, calculation of leukocytes in Goryaev's chamber before and after incubation and further determination of organism sensitivity by leukocytolysis index, different in the fact that in setting of reaction, first, solution of acidin-pepsin is introduced into samples, calculation of leukocytes before incubation is carried out only in control sample, and leukocytolysis index (LCI) is determined after incubation of experimental and control samples with further deduction of percent of spontaneous leukocytolysis (SLC) in control sample and is final value of LCI (3) is to 20%, organism sensitivity is considered to be low, 20-40% - medium, 40% and higher -high.

EFFECT: increase of determination accuracy.

5 tbl, 5 ex

Description

FIELD OF THE INVENTION

The invention relates to veterinary medicine, and in particular to a method for determining the sensitivity of horses to antigens of microorganisms of various taxonomic affiliations, and can be used in clinical practice for the diagnosis of hypersensitivity to microbial antigens.

State of the art

A known method for determining the sensitivity of the animal to tuberculin (in vivo). The method is based on the interaction of the allergen with sensitized T-lymphocytes and antigen-presenting cells (macrophages and Langerhans cells), which causes the release of allergy mediators and the development of a local reaction in sensitized individuals. The staging of skin samples is a diagnostic method for identifying specific sensitization of the body by introducing an allergen under the skin and assessing the magnitude and nature of the inflammatory reaction that has developed (Pat. RU No. 2280872, class G01N 33/68).

The disadvantages of skin testing as a way to determine the sensitivity of the body are as follows: the method is unsafe for the subject, since the occurrence of not only local (local), but also general reactions of the body to the introduction of an allergen, and in case of high sensitivity, the development of severe allergic complications, is not excluded. The reliability of the method is reduced due to positive results after prophylactic or therapeutic administration of vaccines. Sample preparation requires experienced, specially trained personnel who are impeccably knowledgeable in the procedure for their preparation, as well as a constant supply of anti-shock drugs, which necessitates a reaction in a specially equipped room. The expressivity is insufficient, since the results of the sample are taken into account after 24-48 hours and the number of analyzed antigens is limited.

A known method for determining the content of total and allergy-specific IgE by enzyme-linked immunosorbent assay (ELISA). The method is quite sensitive, specific and express (Fradkin V.A. Diagnostic and therapeutic allergens. / V.A. Fradkin. - M .: Medicine, 1990. - 255 p.).

The disadvantage of enzyme immunoassay is the need for special test systems with a specific allergen, but the set of available systems is currently limited. In addition, the method places high demands on the standardization of the test and on the quality of the antigenic components, that is, the reproducibility of the method substantially depends on the quality and manufacturer of the test system.

There is also known a method for determining basophil degranulation (direct and indirect test), based on the manifestation of active degranulation of basophilic white blood cells, sensitized IgE. When a specific antigen is exposed to such cells, they degranulate (Sheley W. Circulating basophile as indicator of hypersensitivity in men / W. Sheley // Arch. Dermal. - 1963. - v. 88. - P.759).

The disadvantage of this method is the complexity of its reproduction, high subjectivity of the results, low expressivity, the presence of laboratory animals in the formulation of an indirect test.

A known method for assessing the presence of hypersensitivity in vitro by recording the activity of sensitized blood cells by specific damage (destruction) of neutrophils (VA Fradkin Diagnosis of allergy by blood neutrophil reactions. / V.A. Fradkin. - M .: Medicine, 1985. - 175 from.).

Closest to the claimed method (prototype) is a method of staging a reaction with blood leukocytes (leukocytolysis), which consists in the following: 0.08 ml of blood and 0.01 ml of specific antigen are added to a test tube with 0.02 ml of a 5% sodium citrate solution (in the control, instead of antigen, the same amount of isotonic sodium chloride solution is added). After careful mixing, the number of leukocytes in each tube is counted in the Goryaev chamber, the contents are incubated for 2 hours at 37 ° C and the number of leukocytes is counted again. The difference in the number of leukocytes before and after incubation in the experiment and control determines the intensity and degree of sensitization to a specific antigen (allergy). The presence of sensitization of the body is detected with a leukocytolysis index of 0.1 and higher (Applied Immunology. / Ed. By A.A.Sokhin, E.F. Chernushenko. - K .: Zdorovya, 1984. - 320 p.).

The disadvantage of this method is the large time spent on counting the number of leukocytes before incubation in experimental samples, it does not have a high reliability of the analysis, the leukocytolysis index established by calculation is not an informative indicator, the number of analyzed antigens is limited.

Disclosure of invention

The objective of the present invention is to develop a method for determining the sensitivity of a horse's organism to antigens of microorganisms of various taxonomic affiliations.

The technical result that can be achieved using the proposed method is to increase the reliability of determining the sensitivity of animals and increase the number of analyzed antigens of microorganisms of different taxonomic affiliations.

с последующим вычетом процента спонтанного лейкоцитолиза (СЛЦ) в контрольной пробе

при значении ПЛЦ до 20% чувствительность организма считают низкой, 20-40% - средней, 40% и более - высокой.The technical result is achieved using a method for determining the sensitivity of horses to antigens of microorganisms of various taxonomic affiliations, including the reaction of leukocytolysis with blood leukocytes, using an antigen, counting the number of leukocytes in the Goryaev chamber before and after incubation, and then determining the sensitivity of the body according to the leukocytolysis index, while first, a solution of acidin-pepsin is introduced into the samples, counting the number of leukocytes before incubation Only in the control sample, as indicator leykotsitoliza (PLC) was determined after incubation of the test and control samples using the formula:

followed by the deduction of the percentage of spontaneous leukocytolysis (FLC) in the control sample

with a PLC value of up to 20%, the sensitivity of the body is considered low, 20-40% - medium, 40% and more - high.

The essence of the method is as follows.

Blood is taken from the animal with the addition of an anticoagulant.

The reaction is put in two test tubes (their number can be increased if more antigens are used), where 0.3 ml of weakly stained with methylene blue solution of acidin-pepsin are added. Then, 0.02 ml of the blood of the test animal is added to each tube. 0.1 ml of Staphylococcus aureus antigen is added to the first test tube (experimental), and 0.1 ml of sterile physiological saline is added to the second (control) one. Samples are thoroughly mixed, the number of leukocytes in the Goryaev’s chamber is counted in a control tube, and the experimental and control samples are placed in a thermostat for 2 hours at a temperature of 37-37.5 ° С.

The antigen contained in the experimental sample causes specific damage to sensitized white blood cells and does not affect white blood cells, which do not show increased sensitivity to it. It is possible to detect leukocyte damage by counting only if they are completely destroyed. The destruction of damaged leukocytes is carried out by the enzyme pepsin, which in an acidic environment acts only on damaged cells and does not act on intact cells. Such selective action of pepsin on damaged white blood cells provides a difference in their calculation.

The experimental and control samples before incubation contain a different number of leukocytes; when antigen is added to the experimental sample, in some cases the reaction is observed immediately, which is confirmed by enhanced leukocyte cytolysis. Counting the number of leukocytes before incubation in the experimental sample leads to a significant waste of time in the analysis of sensitivity to several antigens and does not represent informational value for judging the state of the body.

After incubation, the number of leukocytes in the Goryaev chamber in the experimental and control tubes is counted.

The leukocytolysis indicator for each antigen is determined by the formula:

where PLC is an indicator of leukocytolysis after incubation;

L control - the number of leukocytes in the control after incubation;

L experience - the number of leukocytes in the experience after incubation.

At the same time, the percentage of spontaneous leukocytolysis (FLC) in the control sample is calculated:

where L to. before incubation - the number of leukocytes in the control before incubation;

L to. After incubation - the number of leukocytes in the control after incubation.

The total leukocytolysis value for each antigen is calculated by the formula:

Thus, the determination of the leukocytolysis index to the antigen after incubation of the experimental and control samples in an acidin-pepsin solution, followed by the deduction of the percentage of spontaneous lysis of leukocytes in the control sample, increases the reliability of determining the sensitivity of animals and, reducing the study time, increases the number of analyzed antigens.

Spontaneous leukocytolysis in control samples of the studied animals was observed from 3 to 13% (Table 1-3); therefore, the sensitivity gradation was determined as follows: when the PLC value (3) is up to 20%, a low sensitivity of the organism is concluded, 20-40% - average , 40% or more - high.

The implementation of the invention

Examples of specific performance of the method for determining the sensitivity of horses to antigens of microorganisms of various taxonomic affiliations

Example 1. Stallion Jagan, 8 years old, Akhal-Teke breed. The proposed method determines the sensitivity to antigens Streptococcus faecalis, Staphylococcus aureus, Candida albicans. To do this, take 4 tubes (their number can be increased if more antigens are used), where 0.3 ml of weakly stained with methylene blue solution of acidin-pepsin are added. Then, 0.02 ml of the blood of the test animal with the addition of an anticoagulant is added to each tube. Then, 0.1 ml Streptococcus faecalis, Staphylococcus aureus, Candida albicans antigen, respectively, are added to the first three test tubes (experimental), and 0.1 ml of sterile physiological saline is added to the fourth (control). Samples are thoroughly mixed, the number of leukocytes in the Goryaev’s chamber is counted in a control tube, and experimental and control tubes are placed in a thermostat for 2 hours at a temperature of 37-37.5 ° С. After incubation, the number of leukocytes in the Goryaev chamber in the experimental and control tubes is counted.

The number of blood leukocytes after incubation with the antigens Streptococcus faecalis, Staphylococcus aureus, Candida albicans in the experimental samples was 3200, 3200 and 3650 thousand / μl, respectively, in the control before incubation - 4950 thousand / μl and after incubation - 4300 thousand / μl.

The leukocytolysis rate for Streptococcus faecalis and Staphylococcus aureus antigens (PLC _1.2 ) was 25.6%. The leukocytolysis index for Candida albicans antigen (PLC ₃ ) was 15.1%.

Spontaneous leukocytolysis was 13%:

The total value of PLC for Streptococcus faecalis and Staphylococcus aureus antigens was 12.6%:

PLC _1.2 = 25.6% -13% = 12.6%.

The total value of PLC for Candida albicans antigen was 2.1%:

PLC ₃ = 15.1% -13% = 2.1%.

They conclude that there is a low sensitivity of the body to the antigens Streptococcus faecalis, Staphylococcus aureus, Candida albicans.

Example 2. Stallion Triumph, 8 years old, Kabardian breed. The proposed method determines the sensitivity to similar antigens. The number of blood leukocytes after incubation in experimental samples with Streptococcus faecalis, Staphylococcus aureus and Candida albicans antigens was 7000, 3800 and 2900 thousand / μl, respectively, in the control sample before incubation - 7600 thousand / μl and after incubation - 7000 thousand / μl .

The leukocytolysis rate for Streptococcus faecalis antigen was 0% (PLC ₁ ), for Staphylococcus aureus antigen - 45.7% (PLC ₂ ) and for Candida albicans antigen - 58.6% (PLC ₃ ):

Spontaneous leukocytolysis amounted to 7.8%:

The total value of PLC for Streptococcus faecalis antigen was 0%, for Staphylococcus aureus antigen - 37.9% and for Candida albicans antigen - 50.8%:

PLC ₂ = 45.7% -7.8% = 37.9%;

PLC ₃ = 58.6% -7.8% = 50.8%.

They conclude that there is a low sensitivity of the body to the Streptococcus faecalis antigen, medium sensitivity to the Staphylococcus aureus antigen, and high sensitivity to the Candida albicans antigen.

Example 3. Stallion Grifel, 14 years old, Anglo-Budenn breed. The proposed method determines the sensitivity to antigens Streptococcus faecalis, Staphylococcus aureus, Candida albicans. The number of leukocytes in the experimental samples after incubation was 4300, 3200, 3400 thousand / μl, respectively, in the control sample before incubation - 7000 thousand / μl and after incubation - 6400 thousand / μl. The leukocytolysis rate was 32.8%, 50.0% and 46.8%, respectively. Spontaneous leukocytolysis was 8.5%. The total value of PLC for Streptococcus faecalis antigen was 24.3%, for Staphylococcus aureus antigen - 41.5% and for Candida albicans antigen - 38.3%. A conclusion is drawn on the presence of moderate sensitivity to Streptococcus faecalis, Candida albicans antigens and high sensitivity to Staphylococcus aureus antigen.

Example 4. Stallion Fragile, 10 years old, Trakenninsky breed. The proposed method determines the sensitivity to similar antigens. The number of leukocytes in experimental samples after incubation with Streptococcus faecalis antigen is 2350 thousand / μl, with Staphylococcus aureus antigen - 2400 thousand / μl, Candida albicans - 3000 thousand / μl, in the control sample before incubation - 5000 thousand / μl and after incubation - 4750 thousand / ál. The leukocytolysis rate was 50.5%, 49.5% and 36.8%, respectively. Spontaneous leukocytolysis was 5.0%. The total value of PLC for Streptococcus faecalis antigen was 45.5%, for Staphylococcus aureus antigen - 44.5%, and for Candida albicans antigen - 31.8%, respectively. A conclusion is drawn about the high sensitivity of the body to the antigens Streptococcus faecalis, Staphylococcus aureus and medium sensitivity to the Candida albicans antigen.

Example 5. By the proposed method, the sensitivity of the horses of the equestrian school of the Stavropol GAU to the antigens Staphylococcus aureus, Streptococcus faecalis, Candida albicans (Tables 1, 2, 3) and other antigens of microorganisms of different taxonomic affiliations (Table 4) was determined.

The highest number of animals with high sensitivity (see Table 4) was noted for Staphylococcus aureus antigen, the leukocytolysis rate of which was 46.0 ± 1.3%, and the smallest number of animals - for Escherichia coli and Serratia marcescens antigens, the value of the leukocytolysis index which amounted to 41.3 ± 0.0% and 40.4 ± 0.0%, respectively. The largest number of animals with low sensitivity was observed for the Micrococcus luteus antigen, whose leukocytolysis index was 9.0 ± 2.5%.

Significant differences in leukocytolysis were found in highly sensitive (44.6 ± 1.3%) and low-sensitive (11.4 ± 1.4%) animals (p≤0.001).

The number of animals exhibiting high and low sensitivity was different. The criterion for the divergence of the lambda distributions (λ) was 8.3 (p≤0.001).

The magnitude of the normalized deviation in hypersensitive animals from the average value in the group with a plus sign was 1.3 ± 0.1, and in low-sensitivity animals with a minus sign, 1.2 ± 0.2.

Features of the response of animals to antigens of microorganisms of different taxonomic affiliations and the concentration of total Ig E are presented in Table 5.

Three animals (21%) showed high sensitivity to one antigen (Staphylococcus aureus., See Table 5), 1 (7%) to two antigens (Staphylococcus aureus, Candida albicans), to three or more of the tested antigens ( Streptococcus faecium. Streptococcus faecalis, Staphylococcus aureus, Escherichia coli, Serratia marcescens) - 4 (29%). 6 animals (43%) showed low sensitivity to all antigens.

The results of determining the sensitivity of horses to antigens of microorganisms of various taxonomic affiliations using the leukocytolysis reaction coincide with the data on the content of immunoglobulin E in the blood serum, which is a sensitization marker. So, the IgE concentration in animals hypersensitive to three or more antigens was 507.8 ± 32.2 IU / ml, and in low-sensitivity animals - 294.7 ± 41.1 IU / ml (p≤0.01), while the leukocytolysis rate in animals hypersensitive to three or more antigens was 46.2 ± 0.9%, and in low-sensitivity animals - 13.8 ± 1.1% (p≤0.001). A high level of total IgE in the blood serum of the animal indicates a hypersensitivity state, and the setting of the leukocytolysis reaction determines hypersensitivity to a particular antigen (s) or its absence.

A positive correlation was found between the leukocytolysis index and IgE concentration (r = + 0.8, p≤0.01), the absolute content of lymphocytes (r = + 0.7, p≤0.05), including the T-lymphocyte population (r = +0.8, p≤0.05), T-helpers (r = 0.6, p≤0.05) of basophils (r = + 0.7, p≤0.05) and eosinophils (r = + 0 , 8, p≤0.01) of blood, total serum protein (r = + 0.7, p≤0.01), α-globulins (r = + 0.7, p≤0.01). A negative correlation between the leukocytolysis index was established with the phagocytic activity of neutrophils (r = -0.8, p≤0.01), the phagocytic index (r = -0.7, p≤0.05), and lysozyme activity of blood serum (r = - 0.7; p≤0.05).

Table 1 Leukocytolysis index and horse sensitivity to Staphylococcus aureus antigen No. p / p Animal name Age years The number of leukocytes in the control before incubation, thousand / μl The number of leukocytes after incubation, thousand / μl Spontaneous leukocytolysis,% The indicator of leukocytolysis (final),% Sensitivity in control in experience one Jagan 8 4950 4300 3200 13.0 12.6 low 2 Becket 8 6000 5800 3800 3.0 31.5 average 3 Meleogus 9 5600 5000 2200 10.7 45.3 high four Fragile 10 5000 4750 2400 5,0 44.5 high 5 Breeze 3 11200 10800 4500 3,5 54.8 high 6 Triumph 8 7600 7000 3800 7.8 37.9 average 7 The Dragon fifteen 7100 6600 3500 7.0 39.9 average 8 Danube 17 5500 4900 3600 10.9 15.6 low 9 Azimuth one 7000 6500 3000 7.1 46.7 high 10 Stylus fourteen 7000 6400 3200 8.5 41.5 high

table 2 Leukocytolysis index and horse sensitivity to Streptococcus faecalis antigen No. p / p Animal name Age years The number of leukocytes in the control before incubation, thousand / μl The number of leukocytes after incubation, thousand / μl Spontaneous leukocytolysis,% The indicator of leukocytolysis (final),% Sensitivity in control in experience one Jagan 8 4950 4300 3200 13.0 12.6 low 2 Becket 8 6000 5800 4150 3.0 25,4 average 3 Meleogus 9 5600 5000 2950 10.7 30.3 average four Fragile 10 5000 4750 2350 5,0 45.5 high 5 Breeze 3 11200 10800 5300 3,5 47.4 high 6 Triumph 8 7600 7000 7000 7.8 0 low 7 The Dragon fifteen 7100 6600 3400 7.0 41.5 high 8 Danube 17 5500 4900 2600 10.9 36.0 average 9 Azimuth one 7000 6500 4500 7.1 23.6 average 10 Stylus fourteen 7000 6400 4300 8.5 24.3 average

Table 3 Leukocytolysis index and horse sensitivity to Candida albicans antigen No. p / p Animal name Age years The number of leukocytes in the control before incubation, thousand / μl The number of leukocytes after incubation, thousand / μl Spontaneous leukocytolysis,% The indicator of leukocytolysis (final),% Sensitivity in control in experience one Jagan 8 4950 4300 3650 13.0 2.1 low 2 Becket 8 6000 5800 5100 3.0 9.0 low 3 Meleogus 9 5600 5000 3100 10.7 27.3 average four Fragile 10 5000 4750 3000 5,0 31.8 average 5 Breeze 3 11200 10800 7300 3,5 28.9 average 6 Triumph 8 7600 7000 2900 7.8 50.8 high 7 The Dragon fifteen 7100 6600 3900 7.0 33.9 average 8 Danube 17 5500 4900 2500 10.9 38,0 average 9 Azimuth one 7000 6500 3100 7.1 45,2 high 10 Stylus fourteen 7000 6400 3400 8.5 38,4 average

Table 4 Leukocytolysis and sensitivity of horses to microbial antigens (n = 14) Animals
Antigen Highly sensitive,% (M ± m) Number of animals Low sensitivity,% (M ± m) Number of animals Streptococcus faecium 44.5 ± 2.4 3 14.6 ± 1.6 four Streptococcus faecalis 47.7 ± 1.5 3 13.1 ± 2.8 2 Staphylococcus aureus 46.0 ± 1.3 7 15.4 ± 1.6 2 Micrococcus luteus - 0 9.0 ± 2.5 7 Escherichia coli 41.3 ± 0.0 one 13.8 ± 1.8 four Serratia marcescens 40.4 ± 0.0 one 9.0 ± 3.3 four Candida albicans 48.0 ± 2.8 2 5.4 ± 3.7 2

Table 5 Specificity of animal response to antigens of microorganisms of various taxonomic affiliations and the concentration of total Ig E Groups of animals The distribution of animals by the nature of the response to antigens (number) Leukocytolysis in the group,% (M ± m) one Ig E, IU / ml (M ± m) two Ig E, IU / ml (M ± m) three Ig E, IU / ml (M ± m) Hypersensitive 3 378.8 ± 64.4 one 586.5 ± 0.0 four 507.8 ± 32.2 * 45.9 ± 0.8 ** Low sensitive 6 294.7 ± 41.1 6 294.7 ± 41.1 6 294.7 ± 41.1 13.5 ± 0.9 Note: * p <0.01, ** p≤0.001 compared to low-sensitivity animals

The proposed method in comparison with the prototype and other known methods has the following advantages:

1. The reliability of the analysis is improved by introducing an acidin-pepsin solution into the incubation medium, the selective action of which on damaged leukocytes provides a difference in their calculation, and taking into account the percentage of spontaneous leukocyte lysis in a control sample individually for each animal.

2. Allows you to re-conduct the determination of the sensitivity of the body at various time intervals.

3. Allows you to conduct a differential determination of the sensitivity of the body to microbial antigens of different taxonomic affiliations, which is impossible with the formulation of an intradermal test.

4. It is simple in execution, not laborious, allows to reduce the analysis time in comparison with the known ones.

5. Harmless to animals and performers.

6. Saves expensive reagents and drugs.

Claims (1)

A method for determining the sensitivity of horses to antigens of microorganisms of different taxonomic affiliations, including the reaction of leukocytolysis with blood leukocytes using an antigen, counting the number of leukocytes in the Goryaev chamber before and after incubation, and then determining the sensitivity of the body according to leukocytolysis, characterized in that when setting up the reaction, first samples add a solution of acidin-pepsin, counting the number of leukocytes before incubation is carried out only in the control sample, but for now The leukocytolysis factor (PLC) is determined after incubation of the experimental and control samples according to the formula:

followed by the deduction of the percentage of spontaneous leukocytolysis (FLC) in the control sample

with a total value of PLC (3) up to 20%, the sensitivity of the organism is considered low, 20-40% - medium, 40% and more - high.