Aim. Determine an optimal set of the most effective methods of identification and intraspecies typing of causative agents of glanders and melioidosis. Materials and methods. Bacteriologic, immunochemical, molecular-genetic methods were used. Results. A possibility to identify collection strains of pathogenic and closely related Burkholderia in semiautomatic systems is studied. Means of detection of informative variable genome segments of the specified microorganisms were developed, methods of their genetic typing were selected. Effectiveness of application of precipitating mAbs for differentiation of Burkholderia was established. Data on diagnostic possibilities of immunoglobulins fluorescing based on monoclonal antibodies of various etiotropic directionality for detection and identification of B. mallei and B. pseudomallei are generalized. Experimental series of amplification test-systems for identification of glanders and melioidosis causative agents in real-time PCR format are created. Conclusion. A number of methods for identification and typing of glanders and melioidosis causative agents is proposed.

Studied are immunochemical properties of antigen preparations, the spectrum and molar masses of the components contained. Demonstrated is the significance of vertical SDS-polyacrylamide gel electrophoresis, immunodiffusion test, immunoelectrophoresis assay, rocket immunoelectrophoresis with specific sera for identification and differentiation of Burkholderia. Rocket immunoelectrophoresis should be viewed as the most informative method which allows for differentiation between pathogenic and nonpathogenic for humans Burkholderia under usual terms.

Demostrated is the possibility to differentiate virulent strains of melioidosis and glanders etiological agents from avirulent ones and closely related microorganisms according to the presence of the antigenic complex 8, using rocket immunoelectrophoresis with the serum containing antibodies to this complex.

Summarized are the modern approaches to choosing the methods and techniques of pathogenic burkholderia detection, literary data, and the results of the authors own studies on the problem of development of monoclonal preparations for fast identification of Burkholderia genus representatives. Discussed are the prospects both of modernization of monoclonal preparations and test-systems for detection of glanders and melioidosis etiological agents in different objects and of the differentiation of burkholderia species, and intraspecific typing of melioidosis agent strains.

Objective of the research was to use immunoblotting for studies of epitope targeting in monoclonal antibodies to 200 kDa Burkholderia pseudomallei antigen, which are synthesized by hybridomas-producers from the two collections in the laboratory of immunodiagnostics and biotechnology at the premises of Volgograd Research Anti-Plague Institute. Employed were 8 typical strains of melioidosis agent with the complete antigenic structure. Antigen preparations were separated by means of denaturating vertical electrophoresis in 12 % polyacrylamide gel with 0.1 % sodium dodecylsulfate. During the process of cell-replication, 12 hybridomasproducers were given preparative amounts of monoclonal antibodies to 200 kDa Burkholderia pseudomallei glycoprotein. Following that, immunoperoxidase conjugates were manufactured. Epitope targeting of monoclonal antibodies was evaluated using immunoblotting. With the help of vertical electrophoresis identified was the presence of several mandatory major components contained in the antigen complexes of the salt-water and formamid B. pseudomallei extracts. Differential staining substantiated glycoprotein origin of certain antigen components. Immunoblotting with the stated above antigen preparations revealed epitope targeting of a number of monoclonal antibodies to 200 kDa antigen of melioidosis agent; demonstrated were the differences in their specific interaction with biopolymers which form part of the antigen specter. Those differences were characteristic of hybridomas-producers belonging to different collections, as well as of particular strains of B. pseudomallei.