Siberian Roe Deer (Capreolus pygargus Pallas, 1771) in Ukraine: Analysis of the Mitochondrial and Nuclear DNA

A molecular-genetic analysis of the nucleotide sequences of the cytochrome b gene (1140 base pairs) of the mitochondrial DNA and 17 microsatellite loci of eight samples of roe deer from the Samara forest of Dnipropetrovsk oblast (Ukraine) was carried out. For comparison, 212 corresponding mtDNA sequences of the Siberian and European roe deer and data on the variability of microsatellite markers in 49 representatives of these species were included in the study. It was noted that all the analyzed mitochondrial sequences of individuals from the Samara forest are characteristic of the Siberian roe Capreolus pygargus Pallas, 1771. Four haplotypes were described, all of which belonged to the haplogroup typical for the western part of the range of C. pygargus. A fragment analysis of the microsatellite loci of nuclear DNA confirmed the identification of the investigated group with the Siberian species

CHARACTERISTICS OF THE PROTECTIVE ANTIGEN COMPLEX OBTAINED FROM FRANCISELLA TULARENSIS SSP. NOVICIDA

F. tularensis ssp. novicida, considered earlier as a representative of a separate species, has been recently classed among F. tularensis variety, based on the results of comparative analysis of 16S-ribosomal RNA. Subspecies novicida can cause disease only in immunocompromised humans and is low virulent for rabbits. Despite this, high rate of homology of the nucleotide sequence of F. tularensis intraspecific taxon is established. Objective of the study is to obtain protective surface antigen complex from F. tularensis ssp. novicida Utah 112 (ATTC 15 482) cells and investigate its properties. Materials and methods. Protein, carbohydrate, and lipid content of the antigen preparation was measured using conventional colorimetric methods, SDS-PAGE was conducted according to U.Laemmli, and immunoblotting – to H.Towbin. For purification and molecular mass determination column chromatography was applied. Immune-chromatographic activity was analyzed by immune-enzyme assay. Immunogenicity of the produced preparation was tested on scrub white mice, with LD50 and ED50 calculated according to Karber’s method. Results and conclusions. Carried out has been comparative analysis of physical-chemical, antigenic and bio-chemical peculiarities of the protective antigen complex obtained from F. tularensis Utah 112 cells and equivalent antigen complex obtained from the vaccine strain – F. tularensis 15 NIIEG. Protectivity of the preparation has been tested through inoculation of the immunized white mice with virulent F. tularensis 503/840 strain. Demonstrated have been distinctive features of the new preparation, by structure and composition, as compared to similar antigen from the vaccine producer strain, as well as the slowdown of its immunochemical and protective activities

LIQUID NUTRIENT MEDIUM FOR SUBMERGED CULTIVATION OF TULAREMIA MICROBE

The paper describes an effective liquid nutrient medium, utilization of which in the process of submerged cultivation of the vaccine tularemia microbe strain allows for the production of high concentrations of viable biomass with low rates of dissociation, which is essential in manufacturing of live vaccines. Dry enzymatic hydrolysate of fibrin, by-product of anti-rabies immunoglobulin production is used as a nutrient-rich base of the new nutrient medium

Prospects for Application of Ultrafiltration Technology for the Scaled Preparation of Plague Microbe and Cholera Vibrio Major Antigens

Demonstrated is the possibility of application of ultrafiltration technologies in the process of cholera toxin and plague agent capsular antigen precipitation under production conditions. Application of ultrafiltration techniques permits of the reduction of losses at the stages of isolation and purification of antigen preparations; and concentration of raw material or semi-finished product provides for the reduction of labor inputs. Thus it leads to the increase in productivity and economical efficiency

Development of Food-Raw-Material-Based Nutrient Media for Submerged Cultivation of Cholera Vibrio Strains

Carried out has been comparative assessment between the performances of liquid nutrient pancreatic fibrin overcook-based and bakery yeast autolyzate-based media and conventionally used in manufacturing of cholera vaccine media for submerged cultivation of Vibrio cholerae 569B and V. cholerae M-41 strains. Results of investigation of media quality biological predictors (morphological and biochemical property stability, efficacy of biomass and protective antigen accumulation), which are fibrin hydrolyzate and bakery yeast autolyzate-based, suggest the possibility of using them for the production of cholera vaccine. Deployment of inedible raw material-based media in manufacturing of cholera vaccine is a prospective technology in view of reduction of medical-prophylactic preparation costs. Moreover it allows for solving the problem of protein waste-product disposal, which is generated in the process of anti-rabies immunoglobulin manufacturing, thus decreasing ecological impact on the environment

Improvement of Technology of Cholera Toxin B-Subunit Production

Consideration is given to implementation of state-of-the-art filtration technologies for up-scaled manufacturing of cholera toxin B-subunit, produced by recombinant Vibrio cholerae non O1 KM93 strain. Selected are micro- and ultra-filtration membranes to be incorporated into manufacturing method. Investigated are the properties of cholera toxin B-subunit, obtained applying the pilot technology. The engineered method for up-scaled manufacturing of cholera toxin B-subunit makes the procedure easier-to-maintain due to tangential micro- and ultra-filtration, performed at the stage of purification and concentration. It excludes labor-consuming chromatographic purification, while retaining B-subunit properties. The studies undertaken make it possible to manufacture cholera toxin B-subunit with the same characteristics as in the case of the pilot technology, but under production conditions, and use it as a component for chemical cholera vaccine

Investigating the stability of the Properties of Vibrio cholerae strains – Producers of Active Components of the Chemical Cholera Vaccine

One of the key requirements to producer strains used in the manufacturing of immunobiological preparations is their stability, which consists in maintaining the main cultural, morphological, physiological, and productive properties in a series of generations. This paper describes a comprehensive methodological approach to testing strain stability using in vitro techniques.The purpose of this study was to conduct an integrated analysis of the stability in the strains that produce active components of the chemical cholera vaccine when preparing seed material and at the stage of cultivation.Materials and methods. Toxigenic strains of Vibrio сholerae 569B of the classical biovar, serovar Inaba and V. сholerae M-41 of the classical biovar, serovar Ogawa were used in the work. Cell morphology was monitored through light and transmission electron microscopy. Atomic force microscopy was applied to measure the main parameters of the bacterial cell. The strains were tested for the presence of ctxA gene in the chromosome using the “GenChol” test system with electrophoretic registration of results. Whole genome sequencing of the strains was performed on the Ion Torrent PGM platform using the Ion 318 Chip Kit and the Ion PGM Hi-Q View Chef 400 Kit. To determine the specific activity of cholera toxin and O-antigen, a DOT immunoassay with a conjugate based on staphylococcal protein A and colloidal gold nanoparticles was applied.Results and discussion. The stability of the main properties of industrial V. сholerae strains – producers of the active components of the chemical cholera vaccine has been confirmed using microbiological, immunochemical, molecular-genetic methods and microscopic analysis at all stages of cultivation, and the prospects for using the integrated methodological approach experimentally substantiated. Tailoring of these methods will make it possible to control the stability of producer strains, optimize cultivation conditions and, as a result, increase the yield of the necessary antigenic component of the vaccine

Enhancement of the Technology for Live Tularemia Vaccine Production

Objective of the study was to develop and test new biotechnological approaches for live tularemia vaccine production.Materials and methods: Francisella tularensis 15 NIIEG strain was used as producer-strain; Francisella tularensis 503 strain – as test infecting one. Producer strain was cultivated on solid and liquid nutrient media. Tangential ultrafiltration was performed with the help of microfiltration module “Viva-flow”. Lyophilization was conducted using drying installation – Free Zone 2.5 L.Results and discussion: Application of the designed liquid nutrient medium on the basis of enzymatic fibrin hydrolysate and submerged cultivation of the producer-strain has allowed for a significant biomass yield increment. At the stage of tularemia microbe culture concentration via microfiltration through filtering membranes with pore size of 0.2 μm, in the mode of tangential liquid flow, increased has been the content of microbe cells; the nutrient media residues – removed. Comparative analysis of the obtained in accordance with experimental technique laboratory series of the vaccine and commercial preparation of live tularemia vaccine has demonstrated their conformity with the specific normative properties. It is established that application of modified liquid nutrient medium, submerged cultivation conditions, methods of biomass concentration and separation has no negative influence on the main properties of live tularemia vaccine and will provide for considerable produce-ability increase in the future

Properties of Experimental and Standard Preparations of the Protective O Antigen of <I>Vibrio cholerae</I> M41 Ogawa

Investigated are immunochemical, chemical and biochemical properties of the O-antigen preparations obtained from Vibrio cholerae M41 classical biovar, serotype Ogawa strain using standard manufacturing and improved technologies of their concentrating. The preparations have been concentrated by ultrafiltration in the mode of tangential liquid flow with membranes of various cut-off levels for a particular molar weight. ELISA has revealed equal O-antigen load in all of these preparations. Studies of specific fractures by means of chromatographic and electrophoretic methods have demonstrated their properties to be alike. Chemical composition analysis has also identified coincidence in the load of components under specification. Thus, it has been proved that application of the improved V. cholerae protective antigen concentrating technology for manufacturing cholera chemical bivalent palletized vaccine is justified

EVALUATION OF VIBRIO CHOLERAE 569B INABA PROTECTIVE ANTIGENES, DERIVED ON INDUSTRIAL AND DEVELOPED BIOREACTORS AS WELL AS BY IMPROVED TECHNOLOGY

We evaluated immunochemical, physical and biochemical properties of Vibrio cholerae 569B INABA protective antigenes, derived on industrial and own-developed bioreactors as well as by technology of its concentration by tangential ultrafiltration. We detected, protease, twinase and. lysophospholipase in all samples. Also, dotimmunoanalysis showed equal concentration, of cholerogen-anatoxine and. O-antigen in all samples too. Using chromatography and. electrophoresis, we found their properties as similar. Thus, we suppose to be possible using developed bioreactor as well as technology of Vibrio cholerae 569B INABA protective antigens concentration by tangential concentration during a process of synthetic oral cholera vaccine production