SU1068475A1 - Method for detecting filiform virus of bees - Google Patents

Abstract

METHOD OF INDICATION OF NITEVITNO-GO-BEAN VIRUS, including setting a double immunodiffusion reaction in the test antigen agar with immune serum, in order to increase the effectiveness of the method, use rabbit as immune serum: hyperimmune syvro, rotoku, obtained using a strain of Apis filamentous virus 31.

Description

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The invention relates to veterinary virology, in particular, to the development of a method for indicating the filamentous bee virus (CWP).

CWP causes disease of honeybees, accompanied by loss of ability to flight, pargshichy limbs, the presence of a milky white hemolymph, destruction of hemolymph cells in the bee colony, a constant small death of bees is observed, or seven die. Under modern conditions of development of large beekeeping and mother-farmed state farms, NVP can cause a large economic loss to the national economy.

Morphological signs. NVP particles of ellipsoidal shape, 400-100 nm or 450-150 nm in size, consist of a nucleocapsid, 3000-60 or 3000-40 nm in size, enclosed in a shell.

Physicochemical properties. Nucleocapsid contains a double-stranded DNA with a molecular weight of approximately 12-10 Daltons, floating density in whole particle cesium chloride, nucleocapsid and DNA are 1.28, respectively; 1.36 and 1.71 r / CWP particle contains about 12 proteins with molecular weights from 13 to 70 kDa, which are approximately evenly distributed between the shell, the core and the nucleocapsid.

Biological properties. NVP. affects only honey bees, workers and queens. It is found in the following organs of the affected individuals: brain, ventral nerve, pharyngeal, maxillary and waxy glands, fatty body, large venous gland and reservoir of this gland, midgut, ova, and hemolymph. Reproduction of the virus is noted only in the fatty body and ovaries. In infected cells of the fatty body and ovaries, the destruction of the nuclear membranes is noted. Experimental infection was caused by adult worker bees when fed, and or by injection of vyrussoderzhaschih drugs. Unable to infect bee larvae of the 1st age, mice-suckers, 21-day old people, cockroaches and. larvae of a large wax moth. NVP is widespread in the USA, England, found in Japan, New Zealand and Italy.

NVP was detected by electron microscopic examination of extracts from dead bees or hemo from a live bee 1

The disadvantage of the known display method is that the electron microscope is a unique and expensive equipment and is not available in practical laboratories m.

The purpose of the invention is to increase the efficiency of the method of displaying the filamentous bee virus.

The goal is achieved in that according to the method of indicating the filamentous bee virus, including the formulation of the double immunodiffusion reaction in the test antigen agar with immune serum, rabbit hyperimmune serum obtained using Apis fiPamentous Virus No. 31 is used as the immune serum.

In order to obtain rabbit hyperimmune serum, virions of NVP sh. Tata 1 and ma No. 31 are used as antigens. In the amount of 100-150 pieces infected with this strain and killed by NVP, are mechanically homogenized using the conventional method with the addition of 0.01-0.03 M phosphate buffer pH 7.0-7.2 (1 ml per bee). The homogenate is clarified at 1800-2000 g for 8-10 minutes, the supernatant is centrifuged at 8000-10000 g for 20-25 minutes. The precipitate is suspended in 2-3 ml of 0,010, 03 M phosphate buffer pH 7.0-7.2 and the concentrated vaccinated suspension is centrifuged in a sucrose density gradient from 20 to 60% for 3-3.5 h at 3000032000 d. Collect the fraction, containing a virus, with the help of a suitable syringe with a needle, bring it up to a volume of 3-4.5 ml with physiological saline, divide it into three equal parts, pour into vials and freeze.

Giperimmunizats 1 rabbits. Before the first injection, one part of the resulting antigen is thawed, mixed with a full Freund's adjuvant ratio of 1: 1 and injected into the rabbit in the pads of the hind legs at 0.20, 3 MP, in the femoral muscles at 0.50, 75 ml and intradermally at three points back to 0.2-0.3 ml. A second injection is done after 21 days, with this, one part of the antigen is thawed, brought to a volume of 2-3 ml with physiological saline and injected into the rabbit intravenously. I give the third injection intravenously in the same way 7 days after the second. The exsanguination of rabbits is carried out on the 7-10th day after the third injection, the blood is collected in a sterile flask, held at 3.7 s until a clot forms, separated from the walls of the sterile glass with a sponge and left at night. The next day, the serum is sucked off, preserved with merthiolum in a ratio of 1: 5000-1: 10,000 and sealed in ampoules

The studied bees in the amount of 8-12 pieces are mechanically homogenized with the addition of 8-12 ml of distilled water. Homogenate left

at 1000g2000 g for 8-10 minutes, the supernatant is centrifuged at 8000-10000 g for 20-25 minutes. The precipitate formed is suspended in 0.1-0.2 ml of distilled water and used as an antigen. At the same time, the control positive antigen from bees killed by CGP strain 31 is prepared in the same way. Difco agar is used. The solvent used to prepare a 0.9-1% gel solution is 0.1-0.2 M borate buffer (pH 8.4-8.5) with addition of 5-8 g of sodium chloride per 1 liter of solution. The reaction is placed in Petri dishes or on a slide. Accounting for the reaction is carried out to form precipitation bands.

This method can be presented in two versions: macro method according to Ouchterfoni (1948) and micro method according to Mansi (1958).

Macrohod. In a Petri dish or on a slide, a layer of agar gel 1.5–2 mm thick is obtained; wells with a diameter of 5–7 mm are cut out (the distance between the wells is 3–6 mm). The wells are filled with antiserum and antigens to the brim / incubated in a humid chamber at room temperature. Accounting for the reaction is carried out after 24-36 hours to form a polio precipitation.

Mikromet.od. A layer of agar gel with a thickness of 0.8-1.5 WM (the distance between the wells is 1.41, 6 mm) is obtained in chgi ke Petri or on a glass slide. The wells are filled with antiserum and antigens to the brim, incubated in a humid chamber at 3537 ° C. Consideration of the reaction is carried out after 3-6 hours for the formation of precipitation bands. Using micromethometry and the reaction of double immunodiffusion reduces the consumption of reagents, shortens the time required to obtain the results of the reaction.

Preparation of antigen for reactions. The eight bees studied were mechanically homogenized with the addition of 8 ml of distilled water. The homogenate is clarified at 1000 g for 8 minutes, the supernatant is centrifuged at 8000 g for 20 minutes. The precipitate is suspended

B 0.1 ml of distilled water and used as antigen. In a macro or micro method, dual immunodiffusion reactions with a homologous, antiserum obtained

o antigen forms 1-2 precipitation bands.

The 10 bees under study were mechanized & added with the addition of 10 ml of distilled water. The goat genate was clarified at 1500 g for 9 minutes, the supernatant was centrifuged at 9000 g for 22.5 minutes. The precipitate formed is suspended in 0.15 ml of distilled water and used as an antigen. In the macro- or micro-method of double immunodiffusion reaction with a homologous antiserum, the resulting antigen forms 1-2 precipitation bands.

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The 12 bees studied are mechanically homogenized with the addition of 12 ml of distilled water. The homogenate is clarified at 2000 g for 10 minutes, the supernatant centrifuged at 10,000 g for 25 minutes. The precipitate is suspended in O, 2 ml of distilled water and used as antigen. In the macro- or mnkro5 method of double immunodiffusion reaction with a homologous antiserum, the resulting antigen forms 1-2 precipitation bands.

The proposed method is tested

Q us with a positive result in the laboratory.

The proposed method is simple, accessible and reliable. Its use in veterinary practice will allow you to quickly eradicate this virus. The method will be used in research institutes, regions and district veterinary laboratories x.

Claims (1)

METHOD FOR INDICATING A NITEVITSE BEE VIRUS, including the formulation of a double immunodiffusion reaction on an agar of a test antigen with immune serum, characterized in that, in order to increase the efficiency of the method, rabbit hyperimmune serum obtained using Apis strain is used as an immune serum fi £ amentous virus No. 31. >