Method of diagnostics of leptospirosis in farm animals

FIELD: veterinary medicine.

SUBSTANCE: method of diagnostics of leptospirosis in farm animals, comprising determining the presence of antibodies to antigens of seven serogroups L.hebdomadis, L.pomona, L.tarassovi, L.grippotyphosa, L.canicola, L.sejroe and L.icterohaemorrhagiae in blood serum by the method of enzyme immunoassay, characterised in that the antigen is used as the antigenic proteins of Leptospira of three serogroups mixed in equal quantities - L.tarassovi, L.grippotyphosa and L.hebdomadis. The invention significantly simplifies and minimizes the diagnostics of leptospirosis, enables to implement the operational control of epizootic condition of the animals on leptospirosis.

EFFECT: method has a high specificity, sensitivity and commonality, provides creation of a safe labour conditions for the staff of diagnostic laboratories.

4 tbl, 4 ex

 

The invention relates to veterinary medicine, namely, to the diagnosis of infectious diseases and can be used for the diagnosis of leptospirosis in farm animals.

A known method for the diagnosis of leptospirosis based on using the microagglutination reaction - PMA (GOST 25380-91. Animal agriculture. Methods of laboratory diagnosis of leptospirosis. - Instead of GOST 325386-82; with an introd. 1993-01-01. - M.: Publishing house of standards, 1992. - 30 C.), which is carried out by adding leptospiroses culture to investigated the serum of an animal with subsequent determination of titers leptospirosis antibodies. As antigens for the detection of specific antibodies in the serum of animals in PMA use live cultures leptospires. This is mandatory for use during the planned study on leptospirosis each serum minimum of seven antigens (live Leptospira serogroups L.hebdomadis, L.pomona, L.tarassovi, L.grippotyphosa, L.canicola, L.sejroe, L.icterohaemorrhagiae). Diagnostic strains of Leptospira laboratory support periodic re-seeding every 10-15 days. In the reaction using culture of leptospires in the age of 5-10 days with no signs of agglutination and lysis with the accumulation of microbial cells 50-100 million/cm3.

However, the known method has significant drawbacks: a great complexity, subjective the valuation of diagnostic results, the inability to standardize the diagnosis, necessity of the reaction only under a microscope. Furthermore, the method requires maintaining a stable state of complete diagnostic set of live leptospires, which may pose a threat to employees ' health veterinary laboratories.

For the prototype accepted method of detection leptospirosis antibodies in the serum of pigs using enzyme-linked immunosorbent assay (ELISA), where for the manufacture of antigen used Leptospira 7 serogroups: L.hebdomadis, L.pomona, L.tarassovi, L.grippotyphosa, L.canicola, L.sejroe, L.icterohaemorrhagiae (see RF Patent №2053513, IPC A61K 39/00 from 27.01.96.). However, this method is time consuming, because to obtain the antigen must be used Leptospira 7 serogroups and thus this method is not intended to identify leptospirosis antibodies in other species of farm animals.

The objective of the invention is to expand the Arsenal of methods of diagnosis of leptospirosis, providing in a short time with maximum accuracy and objectivity of the results, indicating the presence of the tested serum samples of farm animals antibodies to pathogenic Leptospira and safe working conditions for personnel diagnostic laboratories.

The problem is solved in that in the method for the diagnosis of mites is Spinoza farm animals, including indirect method of solid-phase ELISA for the detection of antibodies to antigens of seven serogroups L.hebdomadis, L.pomona, L.tarassovi, L.grippotyphosa, L.canicola, L.sejroe and L.icterohaemorrhagiae in serum according to the invention as an antigen used mixed in equal amounts of antigenic proteins from Leptospira three serogroups - L.tarassovi, L.grippotyphosa and L.hebdomadis. This method allows you to identify leptospirosis antibodies in different species of farm animals.

The method is as follows.

The obtained antigen sensibiliser polystyrene tablets for ELISA for 16-18 h at 20-22°C, followed by layering 1% solution of BSA with an exposure of 1 h at 37°C and then triple rinsing.

In two wells contribute 100 ál of positive control sample (K+), in the other two wells, 100 ál of negative control sample ( -). In the remaining wells contribute 50 μl of diluent buffer solution for sera (RDB) and 50 µl sample sera. The solution is stirred for 5 times by pipetting without touching the bottom of the wells.

After making serum samples tablet is placed in a plastic bag or cover with a lid and incubated for 60 minutes at 37°C. After incubation, the wells are exempt from the content and washed five times the work of phosphate-saline buffer solution with twin (FSB-T).

After about Yuki in wells contribute 100 ál of conjugate solution, conjugated to horseradish peroxidase (RKG-G); the tablet is placed in a plastic bag or cover with a lid and incubated for 30 minutes at 37°C.

After the second incubation, the wells are exempt from the content and conduct five wash working solution FSB So After washing the wells contribute 100 μl of a solution of a Chromogen - tetramethylbenzidine substrate (TMB substrate) and incubated for 30 minutes at 37°C in the dark. After this time the reaction is stopped by adding to each well 50 µl of stop solution.

Evaluation of results:

The ELISA results recorded on the spectrophotometer. The optical density (OD) was measured at a wavelength of 450 nm. Zero ("form") set by air. Count Opcrit. according to the formula:

Opcrit.=The PCR-(cf.)+0,2,

where the PCR-(AVG) is the average OD value of the (DIS-) in two wells.

If the value of the optical density of the sample does not exceed Opcit., the samples are considered negative for the presence of leptospirosis of antibodies against serogroups L.pomona, L.tarassovi, L.hebdomadis, L.icterohaemorrhagiae, L.grippotyphosa, L.sejroe and L.canicola.

If the value of the optical density of the sample exceeds Opcit., the samples can be considered positive for the presence of leptospirosis of antibodies against serogroups L.pomona, L.tarassovi, L.hebdomadis, L.icterohaemorrhagiae, L.grippotyphosa, L.sejroe and L.canicola in any combination of the offering.

Example 1. Determined optimal combination of antigenic proteins of different Leptospira serogroups in obtaining antigen.

Used separately antigenic proteins of each of the 5 Leptospira serogroups (antigenic proteins serogroups L.canicola and L.sejroe was not taken for the study, because they have antigenic structure identical with L.icterohaemorrhagiae and L.hebdomadis respectively), and mixed in equal quantities of the antigenic proteins of the seven serogroups.

No. 1 antigenic proteins L.hebdomadis,

No. 2 antigenic proteins L.pomona,

No. 3 antigenic proteins L.tarassovi,

No. 4 antigenic proteins L.icterohaemorrhagiae,

No. 5 antigenic proteins L.grippotyphosa,

No. 6 antigenic proteins L.hebdomadis+L.pomona+L.tarassovi+L.grippotyphosa+L.canicola+L.sejroe+L.icterohaemorrhagiae - polyvalent antigen.

For research ELISA with manufactured antigens used blood serum of rabbits sensitized cultures of leptospires different serogroups. Pre-serum was investigated in the RMA. As a control of specificity used the serum intact rabbits with negative PMA on leptospirosis (table 1).

The results of the studies listed in table 1, show that in the study of blood serum intact rabbits with different antigens in ELISA in all cases, the obtained negative results, which coincided with PMA.

When tested polyvalent antigen (L.hebdomadis+L.pomona+L.tarassovi+L.grippotyphosa+L.canicola+L.sejroe+L.icterohaemorrhagiae) positive readings in ELISA obtained in rabbits sensitized only L.hebdomadis, L.tarassovi, L.canicola and L.sejroe, i.e. not all rabbits.

The maximum number of positive ELISA results in the study of blood serum of rabbits sensitized with each of the antigens seven serogroups obtained using antigenic proteins from L.tarassovi (in rabbits sensitized L.pomona, L.tarassovi, L.canicola, L.icterohaemorrhagiae and L.sejroe). The exception was the blood serum of rabbits sensitized with Leptospira Grippotyphosa and Hebdomadis, which gave ELISA a negative result. It was therefore decided to use for the manufacture of antigen combination of antigenic proteins of Leptospira three serogroups - L.tarassovi, L.grippotyphosa and L.hebdomadis.

Example 2. Test antigen derived from antigenic proteins L.tarassovi, L.grippotyphosa and L.hebdomadis conducted using ELISA in the serum of rabbits, intact and artificially sensitized by leptospirae (table 2).

Table 2
The results of the research on leptospirosis serum of rabbits
№ p/pRabbits The results of the PMAThe results of ELISA
To-0,278
To+the1,916
1Sensitized by leptospiraeGrippotyphosaGrip.1/1002,844
2IcterohaemorrhagiaeIcter. 1/200, Can. 1/1002,329
3PomonaPom. 1/4002,605
4TarassoviTar. 1/2003,049
5HebdomadisHebd. 1/200, Sejro 1/1002,623
6CanicolaCan. 1/1002,227
7SejroeSejr. 1/200, Hebd. 1/2003,186
8a mixture of seven culturesIcter. 1/200, Can. 1/100, Hebd. 1/200,3,328
Pom.1/100, Grip.1/50, Sejr. 1/100,
Tar. 1/200
9The intactnegative0,273

Table 2 shows that the antigen produced using antigenic proteins of Leptospira 3 serogroups - L.tarassovi, L.grippotyphosa and L.hebdomadis revealed in immunoassay analysis of antibody in all rabbits sensitized with leptospirae as each of the seven serogroups, and a mixture of leptospires seven serogroups.

Intact rabbits in ELISA gave a negative result.

Example 3. Developed by antigen in ELISA tested 25 blood serum samples of cattle, some of which were positive for the presence of leptospirosis antibodies according to the results of the reaction microagglutination - PMA (table 3).

Table 3
The results of the research on leptospirosis serum of cattle
№ p/pCattleThe results of the PMAThe results of ELISA
To-0,278
To+the1,916
1areagirls on leptospirosisNegative0,15
2Negative0,345
3Negative0,196
4Negative0,2
5Negative0,197
6Negative 0,186
7Negative0,191
8Negative0,248
9Negative0,268
10Negativeto 0.263
11reacting to leptospirosisHebd. 1/500,77
12Hebd. 1/2000,804
13Tar. 1/1001,23
14Tar. 1/501,065
15Pom. 1/500,649
16Heb. 1/400, Sejr. 1/1000,570
17Sejr. 1/50, Hebd. 1/100, Tar. 1/200 0,779
18Pom. 1/4000,857
19Pom. 1/200, Grip.1/1001,146
20Heb. 1/50, Tar. 1/100, Pom. 1/800, Grip.1/2001,692
21Grip.1/50, Icter. 1/1001,15
22Heb. 1/50, Sejr. 1/50, Tar. 1/50, Pom. 1/100of 1.34
23Heb. 1/50, Sejr. 1/500,609
24Pom. 1/1000,76
25Heb. 1/50, Grip.1/50, Tar. 1/100, Icter. 1/501,097

It was established that all sera positive on the results of the RMA, gave a positive result in ELISA using antigenic proteins of Leptospira 3 serogroups - L.tarassovi, L.grippotyphosa and L.hebdomadis (100% of animals). Reacting negatively in PMA cattle in ELISA were negative (100% of animals).

Example 4. Bit is botany antigen in ELISA tested 14 samples of blood sera of pigs, some of which were positive for the presence of leptospirosis antibodies according to the results of the reaction microagglutination - PMA (table 4).

Table 4
The results of the research on leptospirosis serum of pigs
№ p/pPigThe results of the PMAThe results of ELISA
To-0,278
To+the1,916
1areagirls on leptospirosisnegative0,163
2negative0,387
3negative0,305
4negative 0,266
5negativeof 0.332
6negativeof) 0.157
7reacting to leptospirosisPom.1/500,641
8Pom.1/100to 1.034
9Pom.1/1003,021
10Pom.1/500,763
11Pom.1/4003,417
12Pom.1/2003,224
3Pom.1/400, Icter. 1/8001,214
14Pom.1/800, Icter. 1/1002,137

From table 4 it is seen that all of the blood serum of pigs positive results PMA, gave pological the hydrated result in ELISA using antigenic proteins of Leptospira 3 serogroups - L.tarassovi, L.grippotyphosa and L.hebdomadis (100% of animals). Reacting negatively in PMA pigs in ELISA gave a negative result (100% of animals).

Thus, the proposed method for the diagnosis of leptospirosis in farm animals using the method of indirect enzyme-linked immunosorbent assay with the use of antigenic proteins of Leptospira 3 serogroups - L.tarassovi, L.grippotyphosa and L.hebdomadis greatly simplifies the diagnosis of leptospirosis, due to the use as antigens killed strains of Leptospira, as a result eliminates the difficulties associated with the need to maintain and use in laboratories full diagnostic set of live leptospires. In addition, simplified account of the results obtained reaction, which is carried out on a spectrophotometer, and not in a dark field of view of the microscope, as in the RMA, eliminates the possibility of incorrect interpretation of the results.

This method has the speed of production and accounting, security, and standardization, provides rapid detection of antibodies to any antigen of seven common serogroups pathogen that allows you to quickly and systematically monitor epizootic condition of the animals for leptospirosis.

The proposed method for the diagnosis of leptospirosis allows to diagnose this disease in mi the distribution panel is minimal a short time, it has a high specificity and sensitivity.

Method for the diagnosis of leptospirosis farm animals, including the detection of antibodies to antigens of seven serogroups L.hebdomadis, L.pomona, L.tarassovi, L.grippotyphosa, L.canicola, L.sejroe and L.icterohaemorrhagiae in serum enzyme-linked immunosorbent assay, characterized in that the quality of the antigen used mixed in equal amounts of antigenic proteins from Leptospira three serogroups - L.tarassovi, L.grippotyphosa and L.hebdomadis.



 

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