RU1791454C - Method of mareck disease diagnosis - Google Patents

Abstract

Usage: veterinary virology. SUMMARY OF THE INVENTION: to assess the epizootic situation in poultry farms, poultry is taken from the axillary vein and examined for the presence of antibodies to the myelin basic protein. A positive reaction indicates Marek's disease.

Description

The invention relates to veterinary virology and can be used in poultry farming.

Chicken Marek’s disease is a highly contagious disease caused by group B herpes virus, characterized by the development of lymphoid tumors of the internal organs and lymphoid infiltration of peripheral nerves.

Known methods for diagnosing Marek’s disease based on clinical, episodologic data, pathologic and histological changes, and results of virological studies.

Clinical and epizootological manifestations of Marek’s disease are lesions of the peripheral and central nervous system of the bird, accompanied by lameness, paresis and paralysis of the legs, wings, neck, skin lesions, manifested in the form of thickened follicles of feathers, diffuse tumor-like thickening of the skin, partially with necrosis, eye lesions in the form of pupil deformity with or without discoloration of the iris. However, the appearance of these signs

depends on the degree of damage to the nervous system.

Pathologic histological changes in Marek's disease develop earlier than the external symptoms of the disease manifest and are characterized by lymphoid cell infiltration of the affected organs and tissues. In the peripheral nerves are puffiness, diffuse focal lymphoid cell infiltration of the nerve trunk and its connective tissue membrane, as well as periaxonal demyelination of nerve fibers, on which the neurological symptoms of the disease depend.

However, histopathological studies are laborious and, in addition, require slaughter of the bird, which increases the cost of diagnosis.

The procedure for the virological method for the diagnosis of Marek’s disease, which includes a biological test on chickens, chicken embryos and cell culture, isolation and identification of the virus, as well as antibody identification in a sick and ill bird by means of the diffuse precipitation (RDP) reaction, is long, required

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significant costs for animals and materials.

A known method for diagnosing the early stages of Marek’s disease of chickens, including the introduction of the main protein of myelin into the earring intradermally, reading the skin-allergic reaction after 18-24 hours, as well as histological confirmation of the diagnosis.

However, this method for diagnosing birds is associated with stresses arising from repeated hand picking, allergen injections into the earring, and also when cutting pieces of earrings for histological examination.

The aim of the invention is to reduce labor costs and reduce stress on the bird during the intravital diagnosis of Marek's disease of chickens, increase the efficiency of diagnosis and improve, due to this, the epizootic situation in the poultry industry.

This is achieved by taking 0.5 ml of blood from the axillary vein from the bird to assess the epizootic situation in the poultry industry and testing for the presence of antibodies to the myelin basic protein. A positive reaction indicates Marek's disease.

Example. If Marek's disease is suspected or to assess the epizootic situation, 0.5 ml is taken from the bird. blood from the axillary vein, serum is separated and examined by enzyme-linked immunosorbent assay, which includes the following steps.

1. A specific antigen (the main protein of myelin) is adsorbed on the surface of the polystyrene panel. For this purpose, 0.1 ml of a working solution of the myelin basic protein (diluted 1:20) is added to the wells of the 9b-well panel, incubated for 2 hours at 37 ° C or 16-18 hours at 4 ° C. After that, the unsorbed basic myelin protein is removed and the wells are washed 4-5 times with 0.01 M phosphate-buffered saline (pH 7.2-7.4) containing 0.05% Tween-20 and 0.5 M sodium chloride . After each wash, the inverted panels are dried by tapping on filter paper.

2. 0.1 ml of test serum is added to each well at a 1:50 dilution in wash buffer. In this case, the antibodies present in the samples bind to the antigen (the main myelin protein). Two wells are used to study one sample. A single 96-well panel can be used to examine 45 samples. The remaining 6 wells are used for controls: 0.1 ml are added to two wells

positive serum, in two wells - 0.1 ml of negative serum, in a well designed to control the conjugate, all ingredients are added sequentially

reactions other than serum, instead of which 0.1 ml of wash buffer is added. 0.1 ml of washing buffer was added to the well intended to control the substrate to replace all reaction ingredients, excluding the substrate, and the substrate was added in the same amount. The test and control samples introduced into the wells are incubated for 1.5 hours at 37 ° C. Then the panels are washed 4-5 and dried (p. 1).

5 3, In the wells of the panel, with the exception of the wells, the control of the substrate was added with 0.1 ml of conjugate solution (specific antibodies (anti-species) labeled with peroxidase) diluted with wash buffer up to

0 indicated working dilution that binds to antibodies.

Samples are incubated for 1.5 hours at 37 ° C. At the end of the incubation, the panels are washed 4-5 times and dried,

5 4. 0.1 ml of freshly prepared substrate (0.8 mg / ml of 5-aminosalicylic acid with a content of 0.005% hydrogen peroxide - pH 6.0-6.2) is added to the wells of the panel. Panel with samples left for 30-40 minutes

0 in a dark place, after which the reaction results are taken into account.

The results are taken into account spectrophotometrically at a wavelength of 450 nm, and in the absence of a spectrophotometer, visually.

5 When visually evaluating the results, the color of the test samples is compared with the color of the positive and negative standards.

The reaction is carried out on 4 cross

0 system:

++++ - dark brown staining; +++ - brown staining: ++ - light brown staining; + - yellow staining;

5 - - staining is absent.

The answer is considered positive if the color of the solution in the well with the test sample corresponds to + and above.

The maximum dilution, which still ensures the color of the peroxidase reaction product, is 2.1 times higher than the extinction (optical density) of the solution in wells with negative serum as the serum titer.

5 Solutions in negative serum wells, substrate control, conjugate control should be unpainted. The color intensity in a well with positive serum should be ++++,

 EXAMPLE 2. If Marek's disease is suspected or for assessing the epizootic situation, 0.5 ml of blood is taken from the axillary vein from the bird, the serum is separated and examined at the site of the fluorescent antibodies. The study includes three steps.

1. A specific antigen (the main myelin protein) is sorbed onto a clean, pre-fat-free glass slide that does not have scratches. For this purpose, 3 drops (20 µl each) of the working solution of myelin basic protein are applied at a concentration of 1-2 mg / ml diluted in 0.01 M phosphate-buffered solution (pH 7.2). prepared in a 0.15 M sodium chloride solution. On a glass slide make 3 smears with a diameter of 1.0-1.5 cm and dry them at room temperature.

2. On each smear of the main myelin protein, a drop of the test chicken serum is diluted 1: 4 in phosphate-buffered saline and placed in a moist chamber for 30 min at 37 ° C. Then smears. Wash twice with phosphate buffered saline for 10 minutes and dry at room temperature.

3. On dried smears, 20 µl of conjugate is applied — commercial (produced by the Gamalei Institute of Epidemiology and Microbiology of the Academy of Medical Sciences of the USSR) anti-species gamma globulin labeled with FFITC, in the working dilution indicated on the label. The preparations are incubated in a humid chamber at 37 ° C for 30 minutes, washed 3 times. 5 minutes with phosphate buffered saline, rinsed with distilled water and, after drying in air, examined under a luminescent microscope using a non-luminescent immersion oil or dimethyl phthalate. In this case, primary filters of the SS-4, SS-8, SZS-7 type are used in a microscope in combination with a locking filter of the NS-18 type. Microscopy of stained preparations is carried out in reflected beams incident on an object from above through a microscope objective.

The specificity controls in the study of drugs for the presence of antibodies to the myelin basic protein are:

- positive preparations treated with normal serum in various dilutions;

- positive drugs treated with a luminescent antispecific conjugate without immune unlabeled serum;

- for the specific take the bright green glow of myelin fibers in the form of small granules of various shapes.

In control preparations, specific fluorescence should be completely absent.

The intensity of a specific glow in the preparation is evaluated by a four-point system:

0 ++++ - bright emerald green glow;

+++ - a bright green glow;

++ - a weak green glow;

+ - faint greenish-yellow glow. 5 A positive result indicating a disease of Marek’s disease is considered to be the presence in the preparation of the fibers of the main myelin protein with a luminous intensity of 4 or 3 crosses in 3-5 visual fields. 0 EXAMPLE 3. If Marek's disease is suspected or for assessing the epizootic situation, 0.5 ml of blood is taken from the axillary vein from the bird, serum is separated and examined by latex agglutination reaction, which includes the following steps.

1. A specific antigen (the main myelin protein) is adsorbed onto the surface of polystyrene latex. To this end, 5 parts of the myelin basic protein are taken at a concentration of 16 mg / ml diluted with glycine buffer solution (pH 8.8) and combined with 1 part of 10% domestic carboxyl-containing polystyrene

5 latex with a particle diameter of 0.8 microns. The sensitization of the latex-antigen mixture is carried out for 2 hours in a thermostat at 37 ° C.

After this period, the sensitized latex is precipitated by centrifugation for 10 min at 3000 rpm and resuspended in the same buffer solution as a 1% suspension.

2. The test sera are heated 5 for 20 minutes at 60 ° C or 30 minutes at 56 ° C. Then they are diluted with glycine buffer solution to a working dilution of 1: 4. A drop (0.025 ml) of the diluted serum is applied to the polystyrene 0 plate and a suspension of sensitized latex is added in equal volume. The plate is shaken manually for 2 minutes or placed on the turntable for the same period. Reaction 5 is taken into account after 5-15 minutes. If the serum contains a homologous immune com. Ponent (antibodies to the myelin base protein), the latex particles that stick together form agglutinate. clearly visible to the naked eye. With negative

As a result of the reaction, the latex suspension remains homogeneously turbid, without clumps of agglutinate and bleaching sites.

The latex agglutination reaction is taken into account according to a 3-point system:

+++ - sharply positive reaction: clear agglutination, large flakes in a clear liquid;

++ - positive reaction: agglutination is clearly visible, however, the fund is not completely cleared;

--negative reaction: the liquid remains homogeneously turbid.

The latex agglutination reaction is monitored .........

test chicken serum in the initial dilution (1: 4) + suspension of unsensitized latex (negative reaction); normal chicken serum, not containing antibodies to the myelin basic protein + suspension of sensitized latex: (check for the specificity of the diagnosis); serum from poultry with Marek’s disease (serum homologous) + suspension of sensitized latex (test of diagnosticum activity.

EXAMPLE 4. If Marek's disease is suspected or for assessing the epizootic situation, 0.5 ml of blood is taken from the axillary vein from the bird, the serum is separated and examined by means of a thin-layer immune analysis, which includes the following steps.

1. A specific antigen (the main myelin protein) is sorbed on the surface of a 40 mm diameter polyetheral Petri dish pretreated with ethanol. For this purpose, 2 ml of a working solution of myelin basic protein in a concentration of 1-4 mg / ml prepared in potassium phosphate buffer solution (pH 7.2-7.4) is added to the surface of the cup. The antigen is incubated at 37 ° C for 1 hour or at 4 ° C for 16-18 hours. Then excess antigen is removed and the surface of the dishes is washed 2-3 times with potassium phosphate buffer solution and dried at room temperature. In this case, the same antigen solution can be used 4-6 times. Sensitized Petri dishes are stored until use in a humid chamber at 4 ° C. .

2. On the surface of the plate sensitized with the main myelin protein, 0.025 ml of the test sera are added in a dilution of 1: 4 in a potassium phosphate buffer solution.

On each of the two covers of the Petri dish, 20-30 samples can be examined (each sample is surrounded by a ballpoint pen),

3, The dried cup is set for 1 min in an inverted form over a water bath with a water temperature of 56 ° C,

The results of the reaction are taken into account visually, evaluating the nature of the condensate formed. The condensate formed in areas coated with antigen-antibody complexes looks like large droplets with a diameter of 2-4 mm, and in untreated areas and treated with normal or even immune serum - small drops with a diameter of 0.5-1.0 mm,

PRI me R 5.

1. According to claim 1 of example 3.

2. On the surface of the Petri dish, 2-3 ml of molten 1% agar (temp. 50-56 ° C) prepared in 0.15 M sodium chloride solution with the addition of 1% horse serum is added. After the agar has solidified in it, a hole 3-4 mm in diameter is cut into the hole with the test sera and control sera (normal serum and serum from poultry sick with Marek's disease), 0.025 ml per well. The plate was left for 24 hours at room temperature to diffuse the sera, after which the agar was removed, the plate was washed with potassium phosphate buffer solution.

3. The dried cup is set for 1 min in an inverted form over a water bath with a water temperature of 56 ° C.

The results of the reaction are evaluated visually by the nature of the condensate formed or by the diameter of the zone of large cable condensate, because these values are directly proportional to the concentration of antibodies in the test material, for example, when the concentration of antibodies in the test liquid is 1/4 μg / ml, the diameter of the corresponding zone was 4 mm, at 22 μg / ml - 8 mm, 87.5 mg / ml - 12 mm

The use of a method for the intravital diagnosis of chicken Marek’s disease proposed as an invention allows for the prompt diagnosis of the disease before the development of clinical and episodiological signs. At the same time, there is no need to carry out diagnostics by a method based on reading a skin-allergic reaction, which, due to incomplete detection of a sick bird, is applicable only for group diagnostics, labor costs for diagnostics and stress on the bird are reduced. Due to the above, the diagnostic efficiency is improved and the epizootic situation in the poultry farm is improved.

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Claims (1)

The claims are intended to simplify and speed up the process, in Method for the diagnosis of disease Marekakachestva immunological test using immunological testing test the definition of antibodies to the main white myelin, characterized in that, sku myelin in blood serum.