RU2767688C1 - Method for staging indirect immunofluorescence reaction for diagnosing bovine leukaemia - Google Patents

Abstract

FIELD: veterinary science.

SUBSTANCE: invention relates to veterinary immunology, namely to laboratory research methods, and concerns a method for staging an indirect immunofluorescence reaction for diagnosing bovine leukaemia. Method involves: applying 50 mcl of positive serum on a fixed smear of a lymphocyte suspension recovered by centrifugation on a urografin density gradient; incubation in a humid chamber in thermostat at 37–38 °C for 30 minutes; three times washing with distilled water; drying and applying in a colouring titre of luminescent serum against bovine globulins. Further, the smear is placed in a humid chamber in thermostat for 15–20 minutes; three times washing with distilled water; drying and viewing in a luminescent microscope using a four-cross system for assessing luminescence intensity; reaction control is carried out with negative serum and normal saline.

EFFECT: offered method promotes reduction of examination time and labour costs of a specialist, allows detecting BLV virus at early stages of disease development, and can be used as an alternative method of PCR diagnostics.

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Description

The invention relates to veterinary immunology, namely to laboratory research methods, and can be used to detect virus-infected bovine leukemia virus (BLV) by examining a cell suspension of lymphocytes using an indirect immunofluorescence reaction (IRIF).

Currently, in state programs to combat bovine leukemia, the diagnostics are based on serological research methods - immunodiffusion reaction (RID) and enzyme immunoassay (ELISA); hematological, clinical and pathomorphological methods serve to clarify the diagnosis.

Neither RID nor the more sensitive ELISA diagnostic method in some cases give an adequate answer about infection. So, with the development of the disease in the same animal, the level of antibodies can rise and fall in waves, and in some cases proceed without their formation, which does not allow to fully identify all virus-carrying animals [Krikun V.A. Bovine leukemia and immunological tolerance // Veterinary. - 2002. - No. 6. - S. 7-9.]. In addition, these methods do not distinguish between colostral antibodies and active antibodies produced during viral infection.

Polymerase chain reaction (PCR) is a method with maximum sensitivity and high specificity, which allows more effective detection of FLV-infected animals, as well as to distinguish infected calves from calves with colostral antibodies. However, the widespread use of this method in the diagnosis of leukemia is hampered, first of all, by the high cost of equipment and reagents for PCR.

There is a method of isolation of lymphocytes from the blood of cattle using density gradient centrifugation of urografin [BC Vlasenko, M.A. Bazhin et al. Evaluation of the immune status in cattle with leukemia: guidelines. - Omsk, 2010. - S. 9-10.]. The selection of lymphocytes is carried out as follows: from 76 or 60% urografin using distilled water, a 17% solution is obtained, bringing the density to 1.092 g/cm ³ . Using a syringe with a long needle, 1-2 ml of a 17% solution of urographin is added to Florinsky's test tubes, onto which 1-2 ml of a mixture of equal volumes of blood and Versen's solution are layered, and then centrifuged for 20-30 minutes at 420 g, if the blood is from adult animals, or at 600 g if the blood is from young animals. After centrifugation, lymphocytes are concentrated in the form of a dull disk at the border of two liquids, and granulocytes and erythrocytes settle to the bottom of the tube. The resulting layer of lymphocytes is removed with a syringe with a long needle. The method allows to isolate the lymphoid population of cells from whole blood, providing quick and direct access to the maximum number of cells in the study of their quantitative and functional characteristics under various conditions of the body.

To identify VLKRS, direct and indirect variants of immunofluorescence are also used by examining blood smears and altered organs, short-term and transplanted blood and tissue cultures. Particularly noteworthy is the indirect immunofluorescence reaction (IRIF), which, along with ELISA and polymerase chain reaction (PCR), has become an integral part of the research protocol for most diseases of an infectious nature. In addition to the high sensitivity of the immunofluorescent method, its specificity, versatility, economy, and speed of diagnosis, its important advantage is the possibility of direct pathogen detection, in contrast to indirect, which determines the level of specific antibodies.

A known method of setting RNIF, including the study of short-term and long-lived cultures of leukocytes, the use as antibodies of the serum of a sick cow and labeled with fluorescein-5-isothiocyanate (FITC) rabbit serum against bovine globulins [Syurin V.N., Samuylenko A.Ya., Solovyov B.F., Fomina N.V. Viral diseases of animals. - M.: VNITIBP, 2001. - S. 398]. However, when using leukocyte cultures from leukemic animals, degeneration and death of some of the cells occur, which significantly clogs the preparation with cell decay products.

The closest technical solution to the claimed is a method of setting the reaction [Novikova N.N., Baiseitov S.T., Vlasenko B.C., Krasikov A.P. Application of the reaction of indirect immunofluorescence for the diagnosis of bovine leukemia: guidelines. - Omsk, 2020. - S. 8-9.], which includes applying 50 μl of positive serum to a fixed blood smear (test sample), incubation in a humid chamber in a thermostat at 37-38 ° C for 30 minutes to form an antigen- antibody. After that, the smear is washed three times with distilled water to remove unbound antibodies, dried and luminescent serum against bovine globulins is applied in a coloring titer. Then again placed in a humid chamber in a thermostat at a temperature of 37-38°C for 15-20 minutes. After this time, the smears are washed three times with distilled water, dried and viewed under a fluorescent microscope with mandatory reaction controls (with negative serum and saline).

The indication of the leukemia antigen in blood smears makes it possible to see not only the luminous antigen-antibody complex inside the lymphocytes, but also all the formed elements fixed on the glass. The complexity of taking into account the reaction lies in the fact that in an infected animal, in the absence of lymphocytosis in the field of view of the microscope lens, we can see only erythrocytes or observe from one to three leukocytes, which are represented by both granulocytes and monocytes without a specific luminescent glow. The recommendations on the technique of setting up the reaction indicate that in case of a negative reaction, a smear should be viewed in 30 fields of view of the microscope, which creates an increased burden for the researcher.

The technical result of the invention is to increase the accuracy and reliability, as well as to reduce the time of smear analysis.

The technical solution of the method of setting the reaction of indirect immunofluorescence for the diagnosis of bovine leukemia is: the isolation of lymphocytes from the blood of cattle by centrifugation in a density gradient of a radiopaque substance (urographin). A smear of the “dried drop” type is made from the isolated suspension of lymphocytes, dried, fixed in ethyl alcohol for 20 minutes, then 50 μl of positive serum is applied, incubated in a humid chamber in a thermostat at 37-38 ° C for 30 minutes, washed three times with distilled water, dried and a luminescent serum against bovine globulins is applied in a coloring titer. Then again placed in a humid chamber in a thermostat for 15-20 minutes, washed three times with distilled water, dried and viewed in a fluorescent microscope. The reaction with negative serum and saline is also monitored. To assess the intensity of the glow, a four-cross system is used:

(++++) - very bright luminescence, clearly contrasting against a dark background;

(+++) - bright luminescence;

(++) or (+) - weak glow;

(-) - no luminescence.

A positive result is taken as a specific luminescence of the antigen with a score of four or three crosses.

Distinctive features of the proposed method is that for the preparation of smears, a suspension of lymphocytes is used, isolated by centrifugation on a density gradient of urografin. The proposed method will improve the accuracy and reliability of the method due to the release of ballast elements and the use of a high concentration of lymphocytes in the reaction, in which the bovine leukemia virus is localized, and will also significantly reduce the time spent on smear microscopy.

The essence of the invention is illustrated by specific examples of the method.

Example 1. Study of young cattle

In a farm with a wide spread of leukemia (up to 70% of infected cows), after a preliminary study of the livestock using a polymerase chain reaction (PCR), blood is taken from 33 head of young cattle born to virus-carrying mothers of different ages: up to 1.5 and 3-4 months. Conduct a blood test using RNIF by setting it according to the generally accepted method and the proposed method.

The research results are presented in table 1.

As can be seen from Table 1, in 11 samples of calves under the age of 1.5 months, in which DNA of the bovine leukemia provirus was detected, in 100% of cases a positive reaction was noted in RNIF in smears with cell suspension, while in blood smears only 8 out of 11 goals.

In young animals of 3-4 months of age, a coincidence of the results of PCR with RNIF was established, regardless of the method of its formulation.

With the help of a modified method of setting RNIF in 3 out of 10 (in 30.0% of cases) PCR-negative samples, VLCR was detected and only in 2 by a generally recognized method. A similar picture was observed in the study of blood samples from 3-4-month-old young animals, in which 20 and 10% of VLCR carriers were additionally identified, respectively.

All this testified to the higher accuracy and reliability of the proposed method.

Example 2. Study of cows

Blood is taken from 20 heads of cattle aged 3 to 5 years from a farm with a widespread leukemic infection. As a control, 10 cows of the same age from a farm that is safe for this disease are used. Carry out a diagnostic study of the blood of all animals using the immune diffusion reaction (RID), as well as RNIF by setting it according to the generally accepted method and the proposed method.

The research results are presented in table 2.

As can be seen from Table 2, 12 carriers of VLKRS were identified in the farm (A), which is unfavorable for leukemia (A), using RID. All RID-positive animals also had a positive RNIF both in the reaction with whole blood and with cell suspension.

With the help of RNIF, carried out according to the proposed method, 4 (50%) virus carriers were additionally detected, while only one positive sample was established using blood smears.

It should be noted that in the study of cows from a prosperous farm (B), a negative reaction was noted using both RID and both variants of RNIF.

Thus, the proposed method of setting the RNIF increases the accuracy and reliability, while significantly facilitating the process of interpreting the results during microscopy. Staining lymphocytes without ballast elements, we concentrate the leukemia virus in them. We adjust the microscope lens only for a smear in one field of view and immediately take into account the result by the presence or absence of a specific luminescent glow.

A specialist engaged in microscopy spends 15-30 minutes of working time on visualization of one blood smear, i.e. on average, a minute to view one field of view out of the recommended 30 fields with a negative result. When examining a blood smear without lymphocytosis, even with a positive result for VLKRS, in order to see the antigen in lymphocytes, one has to look through 5 to 10 fields of view in search of a specific luminescent glow, which takes 5-10 minutes or more. The isolation of lymphocytes makes it possible to concentrate the antigen, which can be seen with the help of RNIF in 1-2 fields of view, which reduces the time of luminescence microscopy to a maximum of 2 minutes (7.5-15 times).

Therefore, the proposed method of setting RNIF helps to reduce the time of the study and reduce the labor costs of the work of a specialist, allows you to identify the VLCR virus in the early stages of the development of the disease. Can be used as an alternative method of PCR diagnostics.

Claims (1)

A method for setting up an indirect immunofluorescence reaction for the diagnosis of bovine leukemia, which includes applying 50 μl of positive serum to a fixed smear, incubating in a humid chamber in a thermostat at 37-38°C for 30 minutes, rinsing three times with distilled water, drying and applying luminescent serum in a coloring titer against bovine globulins, re-placing the smear in a humid chamber in a thermostat for 15-20 minutes, rinsing three times with distilled water, drying and viewing in a fluorescent microscope using a four-cross system to assess the intensity of luminescence, control of the reaction is carried out with negative serum and saline, to evaluate intensity of luminescence using a four-cross system, characterized in that a suspension of lymphocytes isolated from the blood by centrifugation on a density gradient of urographin is used to prepare a smear.