RU2518352C1 - Method of integrated assessment of infection level of leukemia virus of herd of cattle - Google Patents

Abstract

FIELD: veterinary medicine.

SUBSTANCE: method of integrated assessment of infection level of leukemia virus of herd of cattle comprises detection of antibodies in the composite sample of milk in enzyme-linked immunoassay. The composite sample of milk is taken from 10 heads, at that an individual sample is taken from an animal in a volume of 40-100 mcL, the composite sample is considered positive which optical signal is three times greater than the signal of the negative control sample. In farms satisfactory in terms of leukemia the composite sample is formed from 20 heads.

EFFECT: expanding the range of methods of diagnostics of cattle leukemia.

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Description

The invention relates to the field of veterinary medicine, can be used to comprehensively assess the level of infection of a herd with cattle leukemia virus (VLCRS infection) during routine health-improving measures, as well as in monitoring healthy and healthy herds.

A known method for detecting antibodies to bovine leukemia virus by enzyme-linked immunosorbent assay (ELISA) in individual samples of serum and milk (Instructions for using the kit to detect antibodies to bovine leukemia virus in blood serum and milk by enzyme-linked immunosorbent assay (Option No. 1 - screening) FSUE Kursk Biofactory and GNU All-Russian Research Institute of Experimental Veterinary Medicine named after Y.R. Kovalenko were developed - Approved by the Deputy Head of the Rosselkhoznadzor on March 10, 201 0 g.).

The disadvantage of this method is its use only with individual samples of biomaterial.

The closest in technical essence to the claimed and adopted as a prototype is a method for detecting antibodies to cattle leukemia virus by enzyme-linked immunosorbent assay (ELISA), in which it is possible to use a combined milk sample (Guidelines for the diagnosis of cattle leukemia. - Moscow, 2000 .).

However, these methodological instructions describe a method for studying individual milk samples and do not provide data for using this method on milk collection samples: the number of individual samples in the collection sample is not determined, the volume of an individual sample and the volume of a collection sample for ELISA, the reaction accounting features are not defined.

The task of the invention is to expand the arsenal of methods for comprehensively assessing the level of infection of a herd with cattle leukemia virus using enzyme-linked immunosorbent assay (ELISA).

The problem is solved in that in a method for comprehensively assessing the level of infection of a herd with cattle leukemia virus, comprising detecting antibodies in a milk assay using an enzyme-linked immunosorbent assay, according to the invention, a milk assay is formed from 10 heads, the volume of milk from individual samples is 40-100 μl, the volume of a pre-assembled milk sample for enzyme-linked immunosorbent assay - 100 μl, consider a positive sample, the optical signal of which exceeds three times the signal of a negative control sample , in prosperous leukemia farms form a combined sample of 20 goals.

The method is as follows.

At the initial stage of the work, the optimal number of individual milk samples used to form 1 precast sample and their volume were determined. For this, samples of milk infected with the cattle leukemia virus (VLCRS) were taken according to the results of ELISA, and according to the instructions, the coefficient (K) was determined.

Samples of milk with a high antibody content had a coefficient (K) of 12.0 ± 3.2, and samples of milk with a low antibody had a coefficient of 3.7 ± 0.8. The coefficient (K) in individual samples was determined in order to establish subsequently how it will change in prefabricated samples - how much to decrease or disappear.

In order to determine the optimal amount of antibody level (by K coefficient) (Tables 1, 2) in a collection milk sample, individual positive samples were diluted with milk from obviously negative animals in ELISA 1: 5, 1:10, 1:20, 1:50 and 1: 100.

To determine the effect of the volume of individual samples on the reaction of a precast milk sample, 20, 40, 100, and 200 μl were taken for each dilution.

Before staging in ELISA, the collection sample was thoroughly mixed and 100 μl was taken for the reaction. An enzyme-linked immunosorbent assay of the collected precast samples was carried out in accordance with the instruction manual.

Table 1 Taking into account the results of ELISA with precast milk samples in various dilutions of samples with a high content of specific antibodies (K coefficient) Breeding
Sample volume, μl 1: 5 1:10 1:20 1:50 1: 100 twenty 7.28 ± 0.43 6.79 ± 0.34 5.40 ± 0.18 3.40 ± 0.13 2.29 ± 0.23 40 7.05 ± 0.35 6.13 ± 0.29 4.82 ± 0.25 3.05 ± 0.26 1.62 ± 0.12 one hundred 6.29 ± 0.26 5.91 ± 0.42 4.43 ± 0.21 2.98 ± 0.18 1.48 ± 0.13 200 6.04 ± 0.20 5.17 ± 0.33 4.00 ± 0.16 2.54 ± 0.15 1.25 ± 0.05

From table 1 it can be seen that the maximum volume and dilution of a precast milk sample, recorded as positive, are 40-100 μl and 1:50.

table 2 Accounting for the results of ELISA with precast milk samples in various dilutions of samples with a low content of specific antibodies (K coefficient) Breeding
Sample volume, μl 1: 5 1:10 1:20 1:50 1: 100 twenty 3.12 ± 0.26 3.27 ± 0.24 1.92 ± 0.16 1.38 ± 0.12 0.31 ± 0.15 40 3.06 ± 0.17 3.15 ± 0.18 1.85 ± 0.11 1.24 ± 0.06 0.22 ± 0.06 one hundred 2.79 ± 0.24 2.95 ± 0.22 1.48 ± 0, S 0.99 ± 0.09 0.18 ± 0.10 200 2.32 ± 0.18 2.01 ± 0.13 1.04 ± 0.08 0.93 ± 0.12 0.11 ± 0.03

Based on the data presented in tables 1 and 2, it was found that as the dilution of positive samples, the optical density signal decreases.

It was found that the optical signal of precast samples three times higher than the signal of the negative control sample can be taken into account as positive, since when individually setting the samples that formed such precast samples, positive milk samples were detected in 100% of cases. When the signal K <3 (K = 2.3 ± 0.29), only in 15% of cases in the collection sample positive individual samples were detected.

As can be seen from table 2 (samples with a low antibody content), the maximum values were V = 100 μl at a dilution of 1:10.

When diluting the samples of milk selected for the experiment 10 times, the reaction of all precast samples was positive. When diluted 20 and 50 times, the results of some samples with a low antibody content were negative or questionable; at a dilution of 1: 100, all samples were negative.

Based on the fact that part of the samples at a dilution of 1:20 and higher yielded a negative result when placed in ELISA, 1:10 was taken for working dilution of precast samples, since during this dilution all samples retained a positive signal.

When conducting diagnostic studies of animals in farms that are prosperous for leukemia, it is advisable to form precast milk samples from 20 animals.

The volumes of individual samples taken for the formation of a precast sample were selected at 40 and 100 μl, since with these volumes of individual samples, the optical signal of the precast sample gives an optimal result when taking into account the reaction.

Example 1. We tested the proposed method for the diagnosis of leukemia in cattle in a production environment. 417 dairy cows from a leukemia-poor household and 352 cows from a leukemia-friendly farm were examined. Collected milk samples in all cases were formed from 10 individual samples. The volume of individual samples for the formation of a precast sample was taken 100 μl. To control from all of these animals, milk was examined individually. The results are presented in tables 3 and 4.

Table 3 Comparative evaluation of the results of ELISA with precast milk samples ELISA with precast samples Control (ELISA with individual samples) Number of samples, pcs. (Number of precast samples, pcs.) 417 (42) 417 The number of positive precast samples. 7 - Total investigated in ELISA, goal. 112 417 In total, infected animals were identified, goal. 12 12

The table shows that in ELISA reacted 7 precast samples. Samples of milk from animals included in these precast samples were examined individually. In this case, 12 positive samples were identified. The results coincided with the benchmarks.

Table 4 Comparative evaluation of the results of IFA diagnostics with prefabricated milk samples in a successful leukemia farm ELISA with precast samples Control (ELISA without precast samples) Number of samples, pcs. (Number of precast samples, pcs.) 352 (35) 352 The number of positive precast samples. 0 - Total investigated in ELISA, goal. 35 352 In total, infected animals were identified, goal. 0 0

The results of studies of precast milk samples from cows of a leukemia-friendly farm coincided with the control, no animals reacting positively in ELISA.

Example 2. We tested the proposed method of setting ELISA with precast milk samples under production conditions with the formation of precast samples from 10 and from 20 animals. 219 milk cows from a successful leukemia farm were examined. The volume of individual samples for the formation of a precast sample was taken 100 μl. To control from all these animals, milk was examined individually in ELISA.

The results are presented in table 5.

Table 5 Comparative evaluation of the results of ELISA with precast milk samples ELISA with precast samples Control (ELISA with individual samples) n is the number of individual samples in the team n = 10 n = 20 Number of samples, pcs. (Number of precast samples, pcs.) 219 (22) 219 (11) 219 The number of positive precast samples. 0 0 - Total investigated in ELISA, goal. 22 eleven 219 In total, infected animals were identified, goal. 0 0 0

From table 5 it can be seen that in prosperous leukemia farms, this research method can be used with the formation of prefabricated milk samples from both 10 and 20 individual samples.

Thus, the study of milk in ELISA in the form of prefabricated samples is not inferior in effectiveness to the standard formulation method, while allowing to optimize the research process. The cost of diagnostic tests is reduced by 2-3 times.

The proposed diagnostic method can be used as an element of the existing systems of health-improving and preventive measures for leukemia, used in the milking herd. And also to control the well-being of dairy herds in healthy and leukemia-friendly farms.

Claims (1)

A method for comprehensively assessing the level of infection of a herd with cattle leukemia virus, including the detection of antibodies in a milk assay in an enzyme-linked immunosorbent assay, characterized in that a milk assay is formed from 10 animals, and an individual animal is taken in an amount of 40-100 μl, positive consider a team sample, the optical signal of which exceeds three times the signal of a negative control sample, in a leukemia-friendly farm, a team sample of 20 goals is formed.