RU2413533C2 - Method for making serum for diagnosing bovine q fever in indirect immunofluorescence test - Google Patents

Abstract

FIELD: medicine, veterinary science.

SUBSTANCE: invention refers to veterinary science. A method involves hyperimmunisation of rabbits with an antigen with scheduled introduction of the immune-stimulating agent levamizole in the first and fourth day of hyperimmunisation, dynamic studying of antibody synthesis on the 7th, 14th, 21st and 30th days, exsanguination of the rabbits and production of serum. Antigen is vaccine strain C.burnetii M-44 inactivated by heating +70° for 30 minutes.

EFFECT: method allows producing Q fever serum with a high diagnostic antibody titres, as well as increasing diagnostic accuracy in bovine Q fever.

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Description

A method for producing coxiellosis serum for the indirect immunofluorescence (RNIF) reaction relates to laboratory methods for the diagnosis of coxiellosis (Q fever) in cattle and can be used in veterinary medicine.

Known methods for producing diagnostic sera for brucellosis, leptospirosis of animals by hyperimmunization of rabbits, used for RNIF, previously described in abstracts of Russian patent documents for 1994-2007 (No. 22257581, 2005.07.27, 95117746, 1997.10.10).

However, the known methods have several disadvantages:

1. Long periods of hyperimmunization.

2. Low titers of antibodies formed in the blood of rabbits.

3. Low activity of the obtained sera.

The closest in technical essence to the claimed method is a method for producing mycoplasmous serum for RNIF by hyperimmunization of rabbits according to the scheme D.Shimmel (1967) in the modification of Novikova N.N. (2002), (Novikova NN Express methods for the diagnosis of associative urogenital urogenital mycoplasmosis of carnivores: Candidate of Science in Science: 16.00.03. / NN Novikova; - Novosibirsk - 2002. - P.55 -60), which is carried out as follows:

1. Three-day cultures of mycoplasmas (M.fermentans, M.canis, M.laidlawii, U.urealyticum) are used for hyperimmunization. The culture fluid is centrifuged at 6 thousand rpm for 1 hour. The precipitate after triple resuspension in physiological saline and subsequent centrifugation was adjusted to a concentration of 1.7 billion microbial bodies in 1 ml according to the bacterial turbidity standard.

2. Hyperimmunization is carried out on 12 rabbits (3 heads for each strain) weighing 2.5-3 kg. The essence of hyperimmunization is the fractional intravenous administration of antigen to rabbits every three days with a double use of the levamisole immunostimulant subcutaneously.

Table 1 The scheme of hyperimmunization of rabbits with mycoplasma cultures The time of administration, days one four 7 10 13 16 twenty 24 Dose of antigen, billion mt 0.85 1.7 1.7 1.7 3.4 3.4 3.4 3.4 Levamisole, ml one one - - - - - -

The dynamics of antibody synthesis is studied on the 7th, 14th, 21st and 30th days after the first injection of antigen in the indirect immunofluorescence reaction, which is set on glass slides according to the standard method, the reaction is recorded in crosses, the glow of less than two crosses is considered negative. In the RNIF, 1 billion suspensions of mycoplasmas inactivated in a water bath at 70 ° C for 30 minutes are used as antigens. On the 30th day, all rabbits are completely bled and receive mycoplasmosis diagnostic sera.

table 2 The dynamics of antibody synthesis in rabbits hyperimmunized cultures Blood collection time (days) Antibody titer 7th day 1:40 14th day 1: 160 21st day 1: 320 30th day 1: 640

The highest level of antibodies is recorded at 21-30 days after hyperimmunization, which ranges from 1: 320-1: 640, respectively (Table 2).

The purpose of our invention is the production of coxiellosis serum for the diagnosis of cattle coxiellosis in the reaction of indirect immunofluorescence by hyperimmunization of rabbits.

The goal is achieved while observing a number of techniques:

1. Obtaining antigen from the vaccine Kuricketsiosis dry live M-44 production NIIEiM them. N.F. Gamalei.

2. Hyperimmunization of rabbits by fractional intravenous administration of antigen every three, four days using the immunostimulant levamisole.

Description of the method of obtaining serum:

For hyperimmunization, an antigen obtained from the vaccine strain C. burnetii M-44 is used. The vaccine was diluted with a standard diluent and warmed up in a water bath at + 70 ° C for 30 minutes in order to inactivate the pathogen.

Then, sterile saline was adjusted to a concentration of 1.7 × 10 ⁹ microbial bodies in 1 ml according to the OSM GISK named after L.A. Tarasevich. Hyperimmunization was carried out on 2 chinchilla rabbits weighing 2.5-3 kg according to the scheme shown in table 3.

Antibody titers were taken into account on days 7, 14, 21, and 30 after administration of the antigen in RNIF, the antigen for which was prepared from the same strain and used after heating in a water bath at 70 ° C for 30 min at a concentration of 0.85 billion mt ., as it was found that at a given concentration of antigen, the reaction is more demonstrative. From the obtained hyperimmune serum, a series of serial two-fold dilutions in physiological saline was prepared, starting at 1:10. Dilutions were made with a microdoser in plastic tablets.

RNIF staging

The indirect immunofluorescence reaction was performed using the following procedure. Several clean drops of antigen were applied parallel to each other on clean, thoroughly degreased, non-scratch slides. Antigens were dried and fixed over the flame of an alcohol lamp. Then, an equal volume drop of serum from each dilution, starting from the last, was applied to each drop of antigen. At the same time they put control:

1) antigen + serum is positive;

2) antigen + serum negative;

3) antigen + saline.

The glasses were kept in a humid chamber at 37-38 ° C for 50-60 minutes to form an antigen-antibody complex. After that, they were washed with distilled water from unbound antibodies, dried and applied in a coloring titer (selected experimentally) a donkey anti-rabbit globulin labeled with FITC. Then the glass was placed for 15-20 minutes in a humid chamber at a temperature of 37-38 ° C. After this time, the smears were washed with distilled water, dried and examined under a luminescent microscope at a magnification of × 1500.

The intensity of the glow was evaluated in crosses:

1) very bright luminescence on the periphery of the microbial cell, clearly contrasting with the cell body - ++++;

2) bright luminescent cell periphery - +++;

3) a weak glow of the periphery of the cell - ++ or +;

4) with a negative reaction, cell luminescence is absent.

For a positive result, a bright specific glow of the contours of microbes in the form of rims with an assessment of 3 and 4 crosses was taken; dubious results were evaluated at 2 crosses in the presence of a weak glow of cell contours.

At the same time, a high level of antibodies was recorded on the 30th day after the start of hyperimmunization, which reached the level of 1: 1280 (Table 4).

Table 4 The dynamics of the synthesis of antibodies in rabbits hyperimmunized with a coxiosis antigen Blood collection time (days) Antibody titer 1 rabbit 2 rabbit 7th day 1:40 1:40 14th day 1: 160 1: 320 21st day 1: 320 1: 640 30th day 1: 1280 1: 1280

Control for the sensitivity, specificity and activity of coxiella serum in RNIF.

To study the sensitivity, species specificity and activity, rabbit coxiella serum was used in dilutions: 1:10, 1:40, 1:80, 1: 160, 1: 320, 1: 640, 1: 1280, 1: 2560, 1: 5120 , coxiellosis antigen obtained by the above method and heterologous antigens (table 5). Serum obtained by hyperimmunization of rabbits with a coxiellosis antigen with a 1:10 dilution was strictly specific for a coxiella antigen with a high level of antibody activity in a titer of 1: 1280.

Table 5 Sensitivity, specificity and activity of coxiella rabbit serum in RNIF Antigens Dilution of coxiella serum 1:10 1:20 1:40 1:80 1: 160 1: 320 1: 320 1: 640 1: 1280 1: 2560 1: 5120 Koksielllesny M-44 + + + + + + + + + - - Mycoplasmosis - - - - - - - - - - - Brucellosis - - - - - - - - - - - Chlamydial - - - - - - - - - - - Listeriosis - - - - - - - - - - - Leptospiral - - - - - - - - - - - IRT-PV - - - - - - - - - - -

The advantage of this method of producing antioxidant serum in higher diagnostic antibody titers and the possibility of using the obtained serum for the diagnosis of cattle coxiellosis in RNIF.

Claims (1)

A method for producing serum for the diagnosis of cattle coxiellosis in an indirect immunofluorescence reaction, including hyperimmunization of rabbits with an antigen provided for by the administration of a levamisole immunostimulator on the first and fourth days of hyperimmunization, studying the dynamics of antibody synthesis on days 7, 14, 21 and 30, bleeding rabbits and obtaining serum, characterized in that the vaccine strain C.burnetii M-44 is inactivated by heating + 70 ° for 30 minutes as an antigen.