RU2366715C1 - Method of immunoelectrophoretic differentiation of melioidosis and fap agents - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: agglutination reaction of immune serum with cells pathogenic burkholderia is carried out. For preparing the immune serum, there are used extracellular antigens (ECA) of typical strain B.pseudomallei C141. ECA is made of elution of bacterial mass grown in a solid nutrient medium, covered with the cellophane, cleaned from cells by centrifugation and filtration. Then ECA is sterilised and separated with bacteriological and biological control of sterility. To prepare the immune serum ECA mixed with Freund's incomplete adjuvant, rabbits are two-cycle immunised. A titre at least 1:32 provides blood sampling and preparing the immune serum. Immunoelectrophoresis (IEP) is performed in agarose plates. ECA of analysed strains B.mallei and B.pseudomallei are introduced in slots with IEP following. Then ECA B.pseudomallei C141 antiserum is introduced in channels. The precipitation lines found in anode and cathode gel regions proves antigens of melioidosis agents to be observed in gel. The absence of the precipitation lines therein specifies FAP agent been introduced in ECA slots. The invention allows differentiating B.pseudomallei and B.mallei, melioidosis and FAP agents being special danger infections without application of the complex equipment.

EFFECT: can be applied as a supplementary diagnostic aid in detection package of melioidosis and FAP agents.

3 cl, 2 dwg, 4 ex

Description

The invention relates to medicine and relates to the differential diagnosis of pathogens of especially dangerous infections of melioidosis (Burkholderia pseudomallei) and glanders (Burkholderia mallei).

Among more than 20 species of the genus Burkholderia, the microorganisms B. pseudomallei and B. mallei have important medical significance, as they are the causative agents of serious diseases of humans and animals - melioidosis and glanders. These bacteria belong to the second group of bacteriological hazards. Despite the differences in the infectious process caused by these microbes, according to many phenotypic characteristics, including antigenic composition, these are very close microorganisms. The proposal has repeatedly been made to consider the glanders pathogen as the biovar of the melioidosis causative agent. Therefore, these bacteria are diagnosed identically until the fact is established that the studied microorganism belongs to the pseudomallei group (it includes pathogens of glanders and melioidosis). Then, to determine the species, tests based on the following phenotypic differences between B. pseudomallei and B.mallei are used: the glancer pathogen is not able to grow on solid nutrient media at a temperature of 42 ° C and when the gentamicin antibiotic concentration in the nutrient medium is more than 4 μg / ml, B. pseudomallei grows under these conditions; the glanders pathogen is motionless, since it does not have flagella, the melioidosis pathogen is mobile, since it has a flagellum (lofotrich) on the cell surface. (Ilyukhin V.I., Popovtseva L.D., Piven N.N. Identification of Pseudomonas pseudomallei // Lab. Business. - 1983. - No. 7. - P.61-62).

Serological reactions (agglutination, complement fixation reaction, indirect hemagglutination reaction), using immune sera to B.pseudomallei and B.mallei cell antigens, have been and are being used in the diagnosis of melioidosis and glanders infections. At the same time, it was not possible to differentiate the causative agents of melioidosis and glanders. The use of the immunoluminescent method based on the obtained monoclonal antibodies against the B.mallei cell antigen made it possible to differentiate the causative agents of melioidosis and glanders (Melioidosis // Collection of scientific works edited by N.G. Tikhonov. / Volgograd: Lower Volga Publishing House, 1995 - 8 -26 p.).

The closest analogue of the proposed method is the use of melioidous antiglutant serum in the agglutination reaction for the differentiation of B. pseudomallei and B.mallei. This method is described in a number of articles by Alekseev V.V., Bondarev I.N., Zykina L.F., which were published in the collection "Issues of anti-epidemic protection" (NIIEM named after N.F. Gamalei. - 1977, vol. 27. - P.54-71) under the title "Indication of the causative agent of melioidosis based on antibodies to the flagellum antigen." To set up the agglutination reaction, the authors applied a rabbit antiserum to the flagellate antigen B. pseudomallei, which was obtained with great labor costs. The specificity of anti-flagellate serum was increased by adsorption of the dried cells of the homologous strain and B.mallei. The production process of the diagnostic drug and the method itself are greatly complicated. However, B. pseudomallei strains without flagella on the cell surface have been described. It is likely that differentiation of the flagella-free B.pseudomallei and B.mallei strains with the above-described anti-flagellate serum will be difficult.

The aim of the invention is to develop a method for differentiating pathogens of melioidosis (B. pseudomallei) and glanders (B.mallei) based on immunoelectrophoresis of their antigens with rabbit immune serum to B.pseudomallei antigens.

This goal is achieved by staging immunoelectrophoresis with extracellular antigens of the analyzed strains of B. pseudomallei and B.mallei and using rabbit antiserum obtained against rabbits against extracellular antigens of a typical B.pseudomallei C141 strain as immune serum.

To obtain immune serum, extracellular (extracellular) antigens (ECAs) of the B. pseudomalei C141 strain are used, which contain the complete composition of antigens with molecular weights of 18, 24, 28 and 34 kDa. ECA is isolated from bacterial washout with a 0.9 M NaCl solution grown for 18 h at 37 ° C after plating on F - agar "Difco", USA, coated with cellophane, daily culture of this strain. Wash off the microbial cells by centrifugation at 8000 g and filtering through a filter with a pore size of 0.45 μm. ECA sterilization is carried out by filtration through a filter with a pore size of 0.22 μm and pouring acetone cooled to -40 ° C (to an acetone concentration of 75% of the total volume). Acetone from the precipitated ECA is removed by centrifugation at 8000 g and drying under traction. The obtained extracellular antigens are subjected to bacteriological and biological control for sterility. Dry ECAs are stored in the refrigerator and used to immunize rabbits. Animals are immunized in two cycles, which include antigen administration four times a month, each of which has a total of 2 ml of a mixture consisting of 1 ml of ECA solution (1 mg / ml) and 1 ml of incomplete Freund's adjuvant, and consists of injections of 0 , 2 ml intradermally paravertebrally at 10 points for each animal with intervals between administrations - 7 days, between cycles - 30 days. Blood is taken from the heart at titer in an immunodiffusion reaction of 1:32 and higher. ECA are used as antigens of the analyzed strain, which are obtained by plating the daily culture of the analyzed strain on agar (F - Difco agar, USA) coated with cellophane, grown for 18 h at 37 ° С, washed with 0.9 M bacterial NaCl solution mass removal from cell washout by centrifugation at 8000 g and filtration through a filter with a pore size of 0.45 μm, precipitation from the washout with simultaneous sterilization of ECA with acetone cooled to -40 ° C (acetone concentration of 75%), removal of acetone by centrifugation at 8000 g for 25 minutes The resulting ECA was dissolved in a 0.9 M NaCl solution, sodium merthiolate was added in a ratio of 1: 10000 and used for analysis. Immunoelectrophoresis (IEF) is carried out in plates of 1% agarose using a veronal-medial buffer with a pH of 8.6. Extracellular antigens of the analyzed B.mallei and B.pseudomallei strains are added to the wells. The IEF is carried out with cooling of the plates for 2.5 hours at a voltage of 4 V / cm. Then, antiserum against ECA B. pseudomallei C141 is added to the trenches. The presence of precipitation lines in the anodic and cathodic regions of the gel indicates the presence of antigens of melioidosis pathogens in the gel. The absence of precipitation lines in similar zones of the gel indicates the introduction of glanders in the ECA well.

Examples of specific performance, confirming the implementation of the proposed method.

Example 1. Obtaining ECA strain B.pseudomallei C141

To obtain extracellular antigens, B. pseudomallei C141 culture was grown on solid nutrient medium (F - Difco agar, USA) at 37 ° C for 18 hours and a suspension of 10 ⁹ microbial cells in 1 ml of a 0.9% NaCl solution was prepared from it pH 7.2. F - agar "Difco", USA sterilely poured into a sterilizer, covered with sterile cellophane and applied to cellophane 8 ml of the previously obtained suspension of bacteria B. pseudomallei C141. The sterilizer is placed for 18 hours in a thermostat with a temperature of 37 ° C. The bacterial mass grown on cellophane is washed off with 32 ml of a 0.9% NaCl solution of pH 7.2, carefully mixed until a homogeneous mixture is obtained and centrifuged for 25 min at 8000 g. Then the supernatant is collected and filtered through a 0.45 μm filter to remove the remaining bacterial cells. The final sterilization is carried out on a filter with a pore size of 0.22 μm and precipitation with acetone cooled to -40 ° C at a final solvent concentration of 75%. Sterility control is carried out by bacteriological and biological methods. The precipitate, represented by extracellular antigens, is freed from acetone by centrifugation for 25 min at 8000 g and drying under traction. The dried filtrates are stored in a refrigerator at + 4 ° C until use.

Example 2. Obtaining immune rabbit serum to extracellular antigens of B. pseudomallei C141 strain.

To obtain hyperimmune serum, rabbits weighing 2-3 kg are used under the age of two years. Animals are immunized over two cycles of ECA administration. For one administration, a mixture of 1 ml of a solution of extracellular B. pseudomallei C141 antigens in 0.9% NaCl, pH 7.2 (concentration 1.0 mg / ml) and 1 ml of Freund's incomplete adjuvant (Difco USA) is used. The immunizing mixture is administered by injection paravertebrally intradermally in 10 points of 0.2 ml. Intervals between administrations are 7 days, between cycles - 30 days. The activity of the sera is determined after each cycle in the RID gel with ECA. Blood is taken at a serum titer> = 1: 32. To suppress the growth of microflora, sodium merthiolate is added to the serum in a ratio of 1: 10000 and 0.2-0.5 ml are ampouled. Then part of the whey is freeze-dried, part is stored in the refrigerator at + 4 ° C.

Example 3. Differentiation of B. pseudomallei and B.mallei in immunoelectrophoresis using antisera against ECA of B. pseudomallei C141 strain.

Immunoelectrophoresis (IEF) is carried out on 8 × 8 cm glass plates pre-coated with an agarose film, which facilitates the pouring of 1% agarose gel on a vertical-medial buffer. Antigens are added to the wells cut in the gel. The main buffer for electrophoresis is Veronal-medinal pH 8.6 buffer with calcium acetate. Glasses with gel are placed in an electrophoresis device on a table cooled to 4 ° C, and bridges of filter paper (6 layers) are applied to the edges of the gel. Bridges are abundantly wetted with buffer. Electrophoresis is carried out at a stabilized current with a voltage gradient in the gel of 4 V / cm. A leading witness (bromphenol blue) is introduced next to the well. Electrophoresis is carried out until the witness moves 2 cm from the center of the glass. After electrophoresis is stopped on an agarose gel, trenches are cut along the direction of electrophoresis into which whole serums are poured. After 12 hours, the glasses are analyzed “under oblique” light. The presence of precipitation lines in the anodic and cathodic regions of the gel indicates the presence of antigens of melioidosis pathogens in the gel. The absence of similar antigens in similar areas of the gel indicates the fact that glanders were added to the well. Immunoelectrophoregrams are presented in figure 1.

Example 4. Characterization of extracellular antigens of B. pseudomallei C141 strain by electrophoretic analysis in SDS page with SDS.

To analyze the composition of extracellular proteins of B. pseudomallei C141 strain, they are separated into fractions by electrophoresis in 10% SDS page with SDS according to Laemmli (1970) in plates measuring 6x8 cm, 0.75 mm thick, using an apparatus for vertical electrophoresis. When preparing samples, dry filtrates were dissolved in a lysing solution of pH 6.8 (0.0625 M Trisaminomethane-Cl, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol) at a concentration of 8 mg / ml, boiled for 10 minutes, centrifuged for 25 minutes at 8000 g. The supernatant is used in experiments. A set of marker proteins from Amersham, USA (molecular weight from 14 to 94 KDa) is used as molecular weight standards. Proteins are stained with Coomassie G-250 and silver (method Oakley et al., 1980). Electrophoregrams are photographed and densitometric. Analysis of the obtained electrophoregrams allows us to establish that the protein spectrum of the extracellular filtrate contains 2 protein fractions stained with Coomassie G-250 with a mol.m. 28 and 34 Sha. When applying the silver staining method, two fractions with a mol.m are detected. 28 and 34 Sha, as in the painting of Coomassie G-250 and two additional fractions with mol. m 18 and 24 Sha (figure 2).

Thus, a method has been developed for differentiating B. pseudomallei and B. mallei, melioidosis and glanders pathogens belonging to especially dangerous infections, by using a new diagnostic drug in immunoelectrophoresis - precipitating antisera against extracellular B. pseudomallei C141 antigens and using ECA as analyzed antigens , which is distinguished by ease of execution, visibility, lack of need for sophisticated equipment. It can be used as an additional diagnostic tool in a set of measures to identify the causative agents of melioidosis and glanders.

The obtained serum gives clearer precipitation lines only with the causative agent of melioidosis both in electrophoresis and in the immunodiffusion reaction.

Claims (3)

1. A method for differentiating pathogens of melioidosis and glanders by immunoelectrophoresis, including the formulation of an agglutination reaction of immune serum with cells of pathogenic burkholderia and the subsequent evaluation of the reaction results, characterized in that extracellular (extracellular) antigens (ECA) of a typical B.pseudalle strain are used to obtain immune serum C141, containing the complete composition of antigens with molecular weights of 18, 24, 28 and 34 kDa, which are isolated from the washout with a 0.9 M NaCl solution of the bacterial mass grown for 18 h at 37 C after plating on agar (F - agar "Difco", USA) coated with cellophane, the daily culture of this strain, the washout is purified from microbial cells by centrifugation at 8000 g and filtration through a filter with a pore size of 0.45 μm, sterilized by filtration through a filter with pore size 0.22 μm and acetone cooled to -40 ° C (final acetone concentration 75%) with simultaneous deposition of ECA, removal of acetone from the precipitate by centrifugation at 8000g and drying of ECA under traction; isolated dried ECA immunize rabbits, and then the resulting immune serum is used in immunoelectrophoresis. 2. The method according to claim 1, characterized in that dried ECA is immunized in two cycles of rabbits, in a volume of 2 ml of a mixture of extracellular antigens of B. pseudomallei C141 in 0.9% NaCl, pH 7.2 (concentration - 1.0 mg / ml) and Freund's incomplete adjuvant ("Difco" USA) in the ratio 1: 1, paravertebrally intradermally in 10 points of 0.2 ml with intervals between administrations of 7 days, and between cycles of 30 days; the titer of the obtained serum should be at least 1:32. 3. The method according to claim 1, characterized in that ECA of the analyzed strains are introduced into the wells made in 1% agarose when immunoelectrophoresis is performed, electrophoresis is carried out for 2.5 hours, at a voltage of 4 V / cm and a stabilized current, then along the direction of the electric current, trenches are cut into which a whole antiserum to ECA B. pseudomallei C141 is added, incubated for 17-20 hours, washed for 1-2 hours on a shaker in a 0.9 M NaCl solution and visual analysis is carried out in an oblique white light immunoelectrophoregram - in this case, in B. pseudomallei strains in the anodic and cathodic zones s at a distance of 3 cm along the trenches with serum precipitin line formed, in B. mallei strains - similar precipitin lines are absent.

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