RU2279088C2 - Method for predicting myxomatosis in rabbits - Google Patents

Abstract

FIELD: laboratory diagnostics.

SUBSTANCE: the present innovation deals with quick test diagnostics of myxomatosis in rabbits. For this purpose it is necessary to carry out specific leukocytic lysis reaction (SLLR) due to applying anti-myxomatosis vaccine out of B-82 strain, calculate SLLR value and at its value being 10% and more myxomatosis should be stated upon. The innovation enables to conduct accelerated diagnostics at earlier stage of the disease mentioned and detect symptom-free flow of myxomatosis, as well.

EFFECT: higher accuracy of diagnostics.

1 cl, 2 ex, 2 tbl

Description

The invention relates to veterinary medicine, in particular to express methods for the diagnosis of myxomatosis.

There are various methods for diagnosing rabbit myxomatosis, including the detection of antigens in the affected skin using fluorescent antibodies, the detection of antibodies, circulating immune complexes using complement fixation reactions, immunodiffusion reactions, neutralization reactions and enzyme-linked immunosorbent assays (Gunenko V.V. et al. diagnosis and prevention of rabbit myxomatosis. Veterinary medicine, 1987, T. 12, S. 44-45). The disadvantages of the known methods are the complexity, the availability of expensive equipment, the inability to determine the presence of the virus in the first days after infection.

The aim of the invention is to reduce the detection time in the body of the animal pathogen myxomatosis. Detection is possible starting from three to six hours after infection.

Achieving this goal is based on the phenomenon of immediate activation, increased sensitivity and overstrain of immunocompetent blood cells - leukocytes - on repeated exposure outside the body of the same antigen that had previously been introduced into the body.

A method for the diagnosis of myxomatosis of rabbits is as follows. A blood sample is taken from the test animal. In a test tube containing 0.05 ml of a 3.8% sodium citrate solution, add 0.1 ml of the test blood and a working dose of antigen of 0.05 ml of live dry culture vaccine against rabbit myxomatosis from strain "B-82" in dilution of one immunizing dose per ml of saline. The control tube is filled in the same volume, but saline is free of antigen.

The tubes are incubated in an incubator for two hours at a temperature of + 37 ° C, shaking every 30 minutes. Then, to destroy red blood cells from the control and experimental tubes, 0.02 ml of blood is transferred to tubes with 0.4 ml of a 3% solution of acetic acid and tinted with a gentian violet. The number of leukocytes in both samples in the Goryaev’s chamber is calculated and the response rate of specific leukocyte lysis (in percent) is calculated by the formula:

where Lk and Lo - the absolute number of leukocytes in the control and experimental samples.

RSLL is considered positive at a rate of 10% or higher.

In the case of severe leukocytosis, when it is difficult to count the leukocytes, the studied control and experimental blood samples are additionally diluted with saline in a ratio of 1: 1 or 1: 2.

Example 1

Ten healthy rabbits were selected to determine the working dose of antigen (vaccine against rabbit myxomatosis of dry live culture from strain "B-82"). Blood from each rabbit was divided into three samples. In three test tubes with 0.05 ml of a 3% sodium citrate solution, 0.1 ml of test blood was added. Then, 0.05 ml of the antigen - vaccine against rabbit myxomatosis of dry live culture from strain "B-82" was diluted in the first test tube in a dilution of four immunizing doses of the drug per ml of physiological solution, and in the second, 0.05 ml of the vaccine in dilution of two immunizing doses per ml of physiological saline, in the third - 0.05 ml of vaccine diluted with one immunizing dose per ml of saline.

The tubes were incubated for two hours at + 37 ° C, shaking every 30 minutes. Then, to destroy red blood cells from control and experimental tubes, 0.02 ml of blood was transferred to tubes with 0.4 ml of a 3% solution of acetic acid and tinted with a gentian violet. Next, we calculated the number of leukocytes in all tubes in the Goryaev chamber and calculated the response rate of specific leukocyte lysis (in percent) according to the formula:

where Lk and Lo - the absolute number of leukocytes in the control and experimental samples.

RSLL for healthy rabbits should be below 10%. The results are presented in table. one.

As can be seen from table 1, the RFLL in all samples did not exceed 10%, therefore, it is more rational to take the dilution of the vaccine against rabbit myxomatosis, dry live culture from strain "B-82" at the rate of one immunizing dose per ml of physiological saline as a working dose of antigen.

Example 2

For a retrospective diagnosis of myxomatosis in a dysfunctional farm, rabbit blood was examined.

Based on the results of the clinical condition of rabbits, 4 groups of animals were formed, 5 animals each (Table 2).

The first group - virus-carrying rabbits, with a hidden form of myxomatosis - were in contact with patients, but without clinical changes. In some, hairless patches were visible, indicating mixed sites in the past.

In the second group - clinically sick with myxomatosis - sick rabbits with swelling of the eyelids, bases of the auricles, anogenetic region, back, with nodular formations in the skin of the ears, head and eyelids were selected.

Rabbits of the third group were vaccinated in order to establish the dynamics of leukocyte lysis from the moment the myxomatous antigen was introduced into the body of the animal, and to identify the peculiarities of the leukocyte lysis reaction in vaccinated compared to virus carriers.

Rabbits of the fourth group are clinically healthy and are not infected with the myxomatosis virus, serve as a control.

In animals of all groups, the response rate of specific leukocyte lysis was determined. For this, blood samples were taken from animals. Each sample was divided into experimental and control. In a test tube containing 0.05 ml of a 3.8% sodium citrate solution, 0.1 ml of the test blood was added and a working dose of antigen of 0.05 ml of the live dry culture vaccine against rabbit myxomatosis from strain "B-82" in dilution of one immunizing dose per ml of saline. The control tube was filled in the same volume, but the saline was free of antigen.

The tubes were incubated in an incubator for two hours at a temperature of + 37 ° C, shaking every 30 minutes. Then, to destroy red blood cells from the control and experimental tubes, 0.02 ml of blood was transferred to tubes with 0.4 ml of a 3% solution of acetic acid and tinted with a gentian violet. We calculated the number of leukocytes in both samples in the Goryaev chamber and calculated the response rate of specific leukocyte lysis (in percent) according to the formula:

where Lk and Lo - the absolute number of leukocytes in the control and experimental samples.

RSLL was considered positive at 10% or higher.

The results are presented in table 2.

table 2
RSLL in rabbits, myxomatosis, vaccinated and healthy Groups of experimental rabbits RSLL, in% Duration of observations in days % Reduction in lysis per day on average (%) at the beginning of the study at the end of the study 1. The latent form, virus carriers. 30-42 29-38 10 0.25 2. Clinically ill with myxomatosis 62-77 60-71 10 0.40 3. Vaccinated with anti-myxomatosis vaccine 29-36 21-28 10 0.80 4. Intact control 4-9 4-9 10 0

As can be seen from table 2, the rate of decrease in the percentage of leukocyte lysis in vaccinated rabbits is 3.2 times higher than in virus-carrying rabbits with a hidden form of myxomatosis, and 2 times more than in clinically ill patients. In patients with the clinical form of myxomatosis, the percentage of leukocyte lysis for the entire observation period was very high, reaching 60-77%.

A sharp increase in the percentage of leukocyte lysis on the third day was found in the group of vaccinated rabbits. This is evidence that the vaccine against myxomatosis of rabbits with dry live culture from strain B-82 causes a sensitization of blood leukocytes (responsible for immunity) before the formation of specific antibodies in the body.

The leukocyte lysis in intact rabbits for the entire period of the experiment did not exceed 10%, i.e. was within normal limits.

The study of RSLL allows you to diagnose the asymptomatic course of myxomatosis.

Claims (2)

1. A method for diagnosing rabbit myxomatosis, characterized in that a specific leukocyte lysis (RSLL) reaction is carried out using an anti-myxomatous vaccine from strain B-82, an RSLL score is calculated and myxomatosis is diagnosed at a value of 10% or more. 2. A method for diagnosing rabbit myxomatosis according to claim 1, characterized in that during leukocytosis, blood samples are diluted with physiological saline in a ratio of 1: 1 or 1: 2 before leukocyte counting.