RU2469321C1 - Method to detect specific antibodies to virus of haemorragic enteritis of turkeys - Google Patents

Abstract

FIELD: veterinary science.

SUBSTANCE: method to detect specific antibodies to a virus of haemorragic enteritis of turkeys consists in survey of a biomaterial from a bird, in a reaction of indirect hemagglutination, where an erythrocytic diagnosticum is represented by erythrocytes of a rabbit with a deposited antigen of the virus of haemorragic enteritis of turkeys, and to control speed of erythrocytes deposition, granules of dextran are used with size from 200 to 500 mcm.

EFFECT: method improvement.

2 tbl, 2 ex

Description

The invention relates to veterinary virology, epizootology, and in particular to methods for detecting specific antibodies [AT] for turkey hemorrhagic enteritis virus [SEI].

A known method for the detection of specific AT to HEI in poultry serum using the enzyme-linked immunosorbent assay [ELISA] (Domeruth, CH, WB Gross and FWPierson. Hemorrhagic enteritis of turkeys. A Laboratory Manual for the Isolation and Identification of Avian Pathogens. 3rd ed. HG Purchase et al. Eds. Kendall / Hunt Publishing Co. Dubuque, IA. Pp. 82-84. 1989), which is an analogue of the proposed method. However, this test does not allow the detection of AT to SEI without the use of commercial test systems related to the species affiliation of the studied samples, and also requires the use of technically sophisticated and expensive equipment to record the reaction results.

The closest in technical terms and adopted for the prototype is a method for detecting AT to the Newcastle disease of birds in the blood serum of a bird using the reaction of delayed hemagglutination [RGCA]. (Czifra G, M Nilsson, DJAlexander, R. Manvell, S. Kecskemeti, BEEngstrom. Detection of PMV-1 specific antibodies with a monoclonal antibody blocking enzyme-linked immunosorbent assay. Avian Pathol. 1996 Dec; 25 (4): 691-703). However, this test does not detect AT to the SEI virus.

The objective of the proposed solution is to expand the arsenal of diagnostic methods for identifying specific antibodies to the SEI virus. The problem is solved in that a method for detecting specific antibodies to turkey hemorrhagic enteritis virus in blood serum or blood plasma of a bird using the indirect hemagglutination reaction [RNGA], characterized in that when preparing an erythrocyte diagnosticum for RNHA, it is necessary to use rabbit erythrocytes deposited on them with the SEI virus antigen, and when setting up the reaction, the indicated erythrocyte diagnosticum, 96-well microplates with a V-shaped bottom and dextran granules from 200 to 500 microns.

The method is as follows.

Materials and equipment: Dindoral turkey hemorrhagic enteritis vaccine, phosphate buffered saline [PBS] pH 7.2-7.4, 1M aqueous alanine solution, buffered 10% formalin solution, centrifuge, shaker.

Protocol for obtaining a diagnosticum.

1. Wash the red blood cells from 2 ml of PBS rabbit blood in a ratio of 1: 5 twice, centrifuging the suspension at 2000 rpm for 1 minute.

2. Resuspend the obtained erythrocyte mass of PBS to 12 ml.

3. Add 1.2 ml of 10% formalin to 12 ml of the suspension.

4. Incubate at 22-24 ° C, gently mixing on a shaker for 2 hours.

5. Precipitate by centrifugation at 2000 rpm for 1 minute.

6. Resuspend the PBS pellet, bringing to a volume of 6 ml.

7. Add 100 μl pre-diluted, in accordance with the instructions and clarified by centrifugation at 10,000 rpm for 2 minutes, the vaccine against hemorrhagic enteritis "Dindoral".

8. Incubate at 22-24 ° C, gently mixing on a shaker for 1 hour.

9. Add 600 μl of a 1M solution of alanine.

10. Incubate at 22-24 ° C, gently mixing on a shaker for 10 minutes.

11. Precipitate by centrifugation at 2000 rpm for 1 minute.

12. Wash the PBS pellet in the ratio 1: 5 once, centrifuging the suspension at 2000 rpm for 1 minute.

13. Resuspend the PBS pellet, bringing the volume to 40 ml.

The resulting erythrocyte suspension is sufficient for 400 samples. The shelf life of the diagnosticum at + 4 ° C is 1 month.

Samples for research: serum samples, blood plasma.

RNGA is carried out according to the following protocol:

1. Prepare granules of dextran with sizes from 200 to 500 microns (commercial preparation - Sefadex G25): resuspend them in PBS 1: 2; incubate for swelling at 22-24 ° C for 2 hours. Use pellet pellet.

2. Prepare serial two-fold dilutions of the test sera starting from a 1: 4 dilution in microplates with a V-shaped bottom in a volume of 100 μl in PBS.

3. Pipette 50 μl Sefadex G25 pellets prepared in accordance with claim 1 into the wells of a 96-well microplate with a V-shaped bottom.

4. Pipette 100 μl of erythrocyte diagnosticum into the wells of the microplate.

5. Incubate at a temperature of 22-24 ° C until the formation of umbrellas for 2-5 hours, you can at night.

In the case of a positive reaction, an umbrella is formed in the thickness of the Sephadex granules, in the case of a negative reaction, red blood cells with the viral antigen immobilized on them freely leak between the granules and settle on the bottom of the V-shaped holes in the form of a button.

Examples are provided to illustrate the method.

Example 1

Results of rnga with blood serum of turkeys of different ages. The dynamics of the growth of antibody titers to the SEI virus is reflected in a bird from a poultry farm that is unfavorable for hemorrhagic enteritis of turkeys.

Table 1 age Total samples neg. 1: 2 1: 4 1: 8 1:16 1:32 1:64 1: 128 1: 256 1 day 12 8 2 2 6 days 12 2 four four one one 10 days 12 four 6 2 13 days 12 6 four 2 18 days 12 2 2 2 2 four 19 days 12 2 2 four 2 2 25 days 12 2 2 6 2 57 days 12 12 122 days 12 12 Note: vaccination with live vaccine against turkey hemorrhagic enteritis was carried out at the age of 22 days.

As follows from the table, the level of antibodies to the vaccination date (22 days) does not decrease due to the natural breakdown of transovarial antibodies, but increases. Thus, one of the leading problems in permanently unfavorable HEI poultry farms is the presence of antibodies before vaccination, which can reduce the effectiveness of vaccination.

Example 2

Antibody titers in RNGA to the HEI virus at the peak of the disease in convalescents.

table 2 GEI virus antibody titers Age 19 days at the peak of the case, number of samples Age 36 days on the decline of the case, the number of samples 0 9 1: 2 1: 4 1: 8 one one 1:16 1:32 9 % reacting (for positive we take 1: 8 and higher) 10% one hundred%

As follows from table 2, protective titers of antibodies in RNGA in the outbreak can be considered the value of 1:32.

Claims (1)

A method for detecting specific antibodies to turkey hemorrhagic enteritis virus, including biomaterial research from poultry, characterized in that the indirect hemagglutination reaction is used, where rabbit erythrocytes with antigen deposited on them are used as an erythrocyte diagnosticum, and erythrocytes are used to adjust the sedimentation rate granules of dextran with sizes from 200 to 500 microns.