RU2754465C1 - Method for obtaining an erythrocyte diagnosticum for the indirect hemagglutination reaction (ihar) with clostridiosis of animals - Google Patents

Abstract

FIELD: biotechnology, veterinary medicine, immunology.SUBSTANCE: a method for obtaining an erythrocyte diagnosticum for the reaction of indirect hemagglutination (IHAR) in animal clostridiosis is proposed, including sensitization of sheep erythrocytes containing formalin and tannin with a mixture of anatoxins of five Cl strains. perfringens No. 28 (type A), LD-1 (type B), No. 392 (type C), No. 213 (type D), No. 342 (type E) in equal proportions obtained from the culture medium by precipitation of formalin-inactivated toxins at a temperature of 37°С (anatoxins) with ammonium sulfate, and somatic antigens obtained by exposure to these Cl. perfringens strains with ultrasound on a disintegrator at a frequency of 20 kHz±500 Hz for 10 min at a temperature of 4°С.EFFECT: provides an increase in the specificity and sensitivity of methods for laboratory diagnostics of anaerobic enterotoxemia (clostridiosis) of unvaccinated livestock of farm animals and control of the intensity of post-vaccination immunity in vaccinated animals.1 cl, 2 tbl, 6 ex

Description

The method of obtaining erythrocyte diagnosticum for the reaction of indirect hemagglutination (RNGA) refers to the methods of laboratory diagnostics of anaerobic enterotoxemia (clostridiosis) of farm animals and can be used in veterinary medicine.

Anaerobic enterotoxemia is an acute disease of animals of various species (sheep, calves, piglets, fur animals, birds, etc.) of the first days of life, characterized by general toxicosis of the body with signs of damage to the nervous system and gastrointestinal tract, resulting from intensive reproduction in the intestines of various types Cl. perfringens and absorption of the resulting toxins [1, 15-17].

The causative agent of the disease is Cl. perfringens, which, according to the release of toxins, are divided into six types: A, B, C, D, E and F, which differ from each other in the antigenic structure of the toxins they produce. In lambs, anaerobic enterotoxemia is caused by the pathogen of types B, D; calves - types A, C, D; in piglets - mainly type C, less often others [2-4, 12-14].

Today, the diagnosis of infectious enterotoxemia in animals is made on the basis of epizootological, clinical, pathological data and the results of laboratory studies [5, 11].

The diagnosis of infectious enterotoxemia of animals is considered established in the case of:

- detection of the toxin in the filtrate of the contents of the small intestine by a biological method and determination of its type in the neutralization reaction with type-specific sera;

- isolation from the contents of the small intestine of a culture producing a toxin, the type of which is established in the neutralization reaction with type-specific sera, with properties characteristic of this pathogen.

The disadvantages of the laboratory method are:

- labor intensity and duration of research, requiring up to 8 working days to make a diagnosis [10];

- high cost of research (high cost of nutrient media, reagents, the cost of purchasing and maintaining laboratory animals).

Also, at present, serological methods (RSK, RDP, RNGA, ELISA, etc.) are used to diagnose many infectious diseases, which make it possible to identify sick animals by titers of specific antibodies in blood serum, as well as to determine the strength of immunity in vaccinated animals. Known methods of obtaining erythrocyte diagnostics used for serological diagnosis of various infectious diseases, such as salmonellosis, mycoplasmosis, escherichiosis [6-8]. In the available sources of scientific and technical literature, no information was found on the method of preparing an erythrocyte diagnosticum for the detection of antibodies to Cl. perfringens.

Closest to the claimed method is a method for producing an enzyme-linked immunosorbent assay for determining specific antibodies to four Cl serotypes. perfringens (A, B, C, D) [9]. However, the disadvantages of the ELISA method are the laboriousness, the duration of the reaction procedure, and the need to purchase expensive equipment.

The technical result to which the invention is directed consists in the development of an indirect hemagglutination reaction (RNGA) for the determination of specific antibodies to Cl bacteria. perfringens, which has high specificity and sensitivity, allowing the simultaneous determination of specific antibodies to five Cl serotypes. perfringens (A, B, C, D, E) and their titers can reliably diagnose anaerobic enterotoxemia among unvaccinated livestock and control the intensity of post-vaccination immunity in vaccinated animals.

To achieve the named technical result, a set of preparations for setting RNGA has been developed, containing a specific erythrocyte antigen produced by sensitizing formalinized and tanned sheep erythrocytes with a mixture of toxoids of five Cl strains. perfringens No. 28 (type A), LD-1 (type B), No. 392 (type C), No. 213 (type D), No. 342 (type E) obtained from the culture medium by precipitation of formalin-inactivated toxins with ammonium sulfate at temperature 37 ° C (toxoid) and somatic antigens obtained by exposure to Cl strains. perfringens No. 28, LD-1, No. 392, No. 213, No. 342 by ultrasound on a Bandelin GM3100 disintegrator at a frequency of 20 kHz ± 500 Hz for 10 minutes at a temperature of 4 ° C, as well as a control positive serum obtained to antigens Cl ... perfringens No. 28, LD-1, No. 392, No. 213, No. 342 with activity in RNGA 1: 256 and control negative serum.

The proposed reaction of indirect hemagglutination is highly sensitive and specific, expressive, does not require expensive equipment, and is easy to set up. The reaction can be delivered even in livestock complexes, which is important when it is necessary to quickly diagnose.

The method of obtaining erythrocyte diagnosticum for RNGA includes:

- obtaining antigens and toxoids Cl. perfringens;

- sensitization of erythrocytes with a mixture of antigens and toxoids;

- obtaining control positive and negative sera.

The inventive method is illustrated by examples of obtaining erythrocyte antigenic diagnosticum.

Example 1. Obtaining antigens necessary for sensitization of erythrocytes from industrial strains Cl. perfringens # 28 (type A), LD-1 (type B), # 392 (type C), # 213 (type D), # 342 (type E). Two types of antigen are obtained - toxoid and somatic antigen.

1.1. Getting toxoids.

Cl toxoids. perfringens types A, B, C, D, E are obtained by inoculating the strains on a meat-liver-casein medium under vaseline oil. Crops are incubated for 8-12 hours at 37-38 ° C. At the same time, the concentration of toxins in the culture reaches up to 4-5 Dlm / cm ³ . To convert toxins into toxoid, formalin is added to the culture to a 0.5% final concentration and kept at a temperature of 37-38 ° C for 10 days. Then, after removing the vaseline oil, the culture is centrifuged at 3000 rpm. within 30 minutes. The sediment, which was the bacterial cells Cl. perfringens are removed, and the toxoid (supernatant) is used to sensitize erythrocytes. Purification and concentration of toxoid is carried out by precipitating it with ammonium sulfate by adding it up to 50% to the total volume (at the final concentration). Then the mixture is centrifuged at 2000 rpm for 30 minutes, then the precipitate is resuspended with distilled water in a ratio of 1:10. The resulting toxoid is dialyzed against distilled water for 24 hours.

1.2. Obtaining somatic antigen.

Strains Cl. perfringens types A, B, C, D, E are plated on mesopatamia agar supplemented with 2% glucose and 15% ram blood. Get a daily culture by cultivation in an anaerostat at a temperature of 37 ° C. The grown culture is washed off with 0.85% isotonic sodium chloride solution. The bacterial suspension is washed three times with sterile saline to free it from the components of the nutrient medium by centrifugation at 2000 rpm for 30 minutes. The sediment of microbial cells is diluted with sterile saline until a suspension of 20 billion microbial cells in 1 ml is obtained according to the turbidity standard. L.A. Tarasevich. Microbial cells are destroyed by ultrasound on a Bandelin GM3100 disintegrator at a frequency of 20 kHz ± 500 Hz for 10 minutes at a temperature of 4 ° C. Then the suspension is centrifuged at 3000 rpm for 30 minutes. The sediment is removed, the supernatant is used as an antigen for sensitizing sheep erythrocytes.

In antigens prepared by methods 1 and 2, the protein concentration is measured using a spectrophotometer according to the Lowry method. The protein concentration is adjusted to 0.4-0.5 mg / ml with phosphate buffered saline (pH 6.4).

Example 2. Preparation of erythrocytes for the load of the antigenic complex.

2.1. Sheep erythrocytes are used as a cellular basis for the preparation of diagnosticum. Blood from donor rams is taken from the jugular vein, defibrinated according to the generally accepted method and diluted 1: 1 with phosphate buffered saline (pH 6.4).

A 20% formalin solution is added to 100 ml of diluted blood in portions in increasing volumes every 15 minutes: 6, 7, 8, 9 and 10 ml. The mixture is incubated in a water bath, stirring thoroughly after each addition. Then the mixture is centrifuged at 1500 rpm for 15 minutes, washed three times with phosphate-buffered saline (pH 7.2) and a 3% suspension of erythrocytes is prepared. The erythrocyte suspension is defended at a temperature of (4 ± 2) ° С for 2 days.

2.2. For the subsequent modification of the erythrocyte surface, an equal volume of tannin solution (1: 20000) in physiological saline is added to the resulting suspension. The mixture is incubated at a temperature of (37 ± 1) ° C on a magnetic stirrer for 20 minutes, erythrocytes are precipitated by centrifugation at 1500 rpm. The erythrocyte sediment is washed three times with phosphate-buffered saline (pH 7.2) and resuspended in the same solution to 3% erythrocyte concentration.

2.3. For sensitization of erythrocytes, a mixture of toxoids and somatic antigens of five Cl serotypes is used. perfringens (A, B, C, D, E) combined in equal volumes. An equal volume of the composite antigen mixture is added to a 3% suspension of tanned erythrocytes. The suspension is incubated in a water bath at a temperature of (37 ± 1) ° С with continuous stirring for 2 hours. Then the sensitized erythrocytes are washed three times with phosphate-saline buffer solution (pH 7.2) by centrifugation at 1500 rpm and resuspended in the same solution to 3% concentration. The resulting erythrocyte diagnosticum is preserved with formalin up to 1% of the final concentration.

Example 3. Obtaining control sera.

3.1. To obtain a positive control serum to Cl antigens. perfringens types A, B, C, D, E, ten rabbits weighing 3.0-3.5 kg are used. For hyperimmunization, the composite mixture of antigens is injected into the marginal ear vein three times with an interval of 4 days. At the first stage, the volume of the injected mixture is 0.5 ml, then 1 ml and 2 ml. Seven days after immunization, blood is drawn to check the titers and specificity of the antibodies present.

3.2. Negative control serum is obtained from rabbits aged from six months to one year, after keeping them in quarantine for 30 days. Preliminary, blood serum is examined in ELISA for the content of specific antibodies.

Example 4. Determination of the activity and specificity of erythrocyte antigenic diagnosticum.

4.1. To determine the activity and specificity, RNGA is performed with control sera (positive and negative to Cl. Perfringens) and heterologous (hyperimmune sera to Escherichia coli, Moraxella bovis). The reaction is set up in round-bottom plates for immunological reactions with a volume of 200 μl.

Control sera are diluted from 1: 8 to 1: 1024. A 0.15 M phosphate buffered saline solution, pH 7.2-7.4, is used as a diluent. Three series of erythrocyte clostridial diagnosticum were tested in RNGA, the results of which are shown in Table 1.

The table shows that with a positive Clostridial serum, the diagnosticum reacts to four crosses up to a dilution of 1: 128, to three crosses 1: 256, to one cross 1: 512. With heterologous sera, a dubious response to two crosses is observed at a 1: 8 dilution. With negative serum and phosphate buffered saline (pH 7.2), a negative reaction is observed in all dilutions.

Example 5. Illustration of the use of RNGA.

The ampoule with the erythrocyte antigen is thoroughly shaken until a homogeneous suspension is obtained, opened and the antigen is diluted in a 1: 2 ratio with a diluent. A 0.15 M phosphate buffered saline solution, pH 7.2-7.4, is used as a diluent.

Preparation of 0.15 M phosphate buffered saline. Into a 1 L volumetric flask, add 20.4 g of chemically pure anhydrous monosubstituted potassium phosphate, dissolve in a small amount of distilled water and bring up to the mark of 1 L with water (solution A). In another 1 liter volumetric flask, 26.7 chemically pure disubstituted sodium phosphate dihydrate is added, dissolved in a small amount of water and brought to the mark of 1 liter with water (solution B). Solutions A and B can be stored at 4-6 ° C for a month.

To prepare a buffer, 76 ml of solution B is added to 24 ml of solution A and 0.85 g of chemically pure sodium chloride is added. Using a pH meter, check the pH of the buffer and, if necessary, adjust the pH to 7.2-7.4 with solutions A or B.

Statement of the reaction.

RNGA is set using microtiter plates with U-shaped wells of Takachi or Tittek microtiter kits. Add 0.05 ml of diluent to the wells of the micropanel using a multichannel pipette. In the first wells with a single-channel pipette, the test sera are added in an amount of 0.05 ml, and by successive transfers, two-fold dilutions of the sera from 1: 2 to 1: 256 are obtained. Then, 0.025 ml of 1% suspension of erythrocyte antigen is added to all wells. The panel is gently shaken and left at room temperature for 6 hours. Simultaneously with the RNGA, controls are set: control for the absence of spontaneous agglutination of erythrocyte antigen in the diluent. In 4-6 wells of the micropanel add 0.05 ml of diluent, then 0.025 ml of 1% suspension of erythrocyte antigen; control for the absence of agglutination of erythrocyte antigen by negative serum of cattle. 0.05 ml of diluent is added to a row of micropanel wells, 0.05 ml of negative serum is added to the first well and titrated as test sera, then 0.025 ml of 1% suspension of erythrocyte antigen is added to each well; control for the activity of erythrocyte antigen with specific serum of cattle. 0.05 ml of diluent is added to a row of micropanel holes, 0.05 ml of specific serum is added to the first well and titrated as test sera, 0.025 ml of 1% suspension of erythrocyte antigen.

Taking into account the reaction.

The reaction is taken into account after 2-2.5 hours. The highest dilution of serum, which gives a clear agglutination (umbrella), is taken for the titer in the RNGA.

Accounting for the reaction is carried out only under the condition of clear results obtained in the controls.

Reaction control.

In the control wells for spontaneous agglutination, erythrocytes should sit in the form of a button or a tight ring.

The negative serum titer should not exceed 1: 4.

The specific serum titer must be at least 1: 128.

The results of RNGA with the tested sera are assessed by titers: positive - 1:16 and higher, doubtful - 1: 8, negative - 1: 4, 1: 2 and zero.

Example 6. Testing RNGA in production conditions.

In the production test of the effectiveness of the kit, blood samples of cattle were used, delivered from cattle farms of the Republic of Tatarstan, different in epizootic terms for clostridiosis. At the same time, blood sera from sick, unvaccinated and healthy, vaccinated animals from farms with clostridiosis dysfunctional, as well as from intact animals from safe farms were studied. The results of the study of blood sera are presented in table 2.

The table shows that RNGA has a high specificity. It allows you to determine specific antibodies to Cl bacteria. perfringens in 96.9% of healthy vaccinated animals and 92.7% of animals with anaerobic enterotoxemia.

At the same time, it was found that the components of the kit do not react with blood sera obtained from intact animals from farms without anaerobic enterotoxemia.

Thus, RNGA makes it possible to identify sick animals among unvaccinated livestock by the titers of specific antibodies in blood serum, as well as to determine the strength of immunity in vaccinated animals.

Sources of information

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Claims (1)

A method of obtaining an erythrocyte diagnosticum for the reaction of indirect hemagglutination (RNGA) in clostridiosis of animals, characterized in that the erythrocyte diagnosticum is made by sensitizing formalinized and tanned sheep erythrocytes with a mixture of toxoids of five strains of Cl. perfringens No. 28 (type A), LD-1 (type B), No. 392 (type C), No. 213 (type D), No. 342 (type E) in equal proportions obtained from the culture medium by precipitation of formalin-inactivated toxins at temperature of 37 ° C (anatoxins) with ammonium sulfate, and somatic antigens obtained by exposure to Cl strains. perfringens No. 28, LD-1, No. 392, No. 213, No. 342 by ultrasound on a disintegrator at a frequency of 20 kHz ± 500 Hz for 10 minutes at a temperature of 4 ° C.