Analysis of the Modern Methods and Means for Rapid Toxin Indication

The risk of occurrence of emergency situations different in their character is still a pressing issue for all the states of the world's community, including the Russian Federation. Nowadays, development of the modern methods and tools for rapid toxin detection and identification holds a high position in biological safety system. Specified means for biological toxin detection do not comply with the requirements of rapidness. One of the prospective means for rapid toxin indication is immune-chromatographic, immunochip, and immunosensor test-systems with high sensitivity, differentiation capacity and expression. In this review described are some up-to-date foreign and home-grown technologies for rapid toxin indication

Ethical Principles for Handling Laboratory Animals in an Experiment with Pathogenic Biological Agents of the I-II Groups

Current international and major part of national documents, regulating experiments on laboratory animals, is set out in Bioethics Convention 3”R”. Presently this 3”R” principle is still relevant and commonly accepted global standard. Objective of the study was to verify the compliance of the staff at the institute with the ethical principles of biological safety when handling laboratory animals infected with pathogenic biological agents (PBA) of the I-II groups, performing diagnostic, experimental, and manufacturing activities. Results and discussion. Carried out analysis of ethical principle implementation, when working with biomodels infected with pathogenic biological agents of the I-II groups, performing scientific and manufacturing activities shows that operational procedures comply with the requirements of biological safety and regulations of ethical code, as well as international standards for humane treatment to animals in biomedical investigations. Conclusions. The work with laboratory animals infected with PBA of the I-II groups at the premises of RusRAPI “Microbe” meets the requirements of biological safety and norms of ethical code. Experiments on animals are aimed at obtainment of new scientific knowledge, preservation and improvement of human health, and also monitoring of natural foci of particularly dangerous infections following the rules of 3“R” bioethical concept

Efficiency of Using the Hydrogen Peroxide Vapor Generator “Fhileas 75” for Disinfection of the Air Ducts of separately Ventilated system for Infected Animal Housing

The aim of the work was to evaluate the efficiency of using the “Fhileas 75” hydrogen peroxide vapor generator for decontaminating the air ducts of the individually ventilated system, “Bio A.S.”, for housing of infected animals.Materials and methods. The hydrogen peroxide vapor generator “Fhileas 75” (France), a disinfectant manufactured by “FHILEASAFE” (7 % hydrogen peroxide solution and 0.15 % peracetic acid solution), separately ventilated system “Bio A.S.” (Germany) for the infected animal housing were applied in the work. Serratia marcescens 9 was used as test-culture.Results and discussion. The efficiency of using the hydrogen peroxide vapor generator “Fhileas 75” for decontamination of air ducts and internal surfaces of the rack of the individually ventilated system “Bio A.S.” on the test-culture S. marcescens 9 at 1·106 mc/ml concentration has been established (operation parameters of the individually ventilated system unit are as follows: air exchange rate – 60 changes per hour, air flow volume – 28 m3/hour, number of disinfection cycles – 5, disinfectant spraying time – 97 min, exposure time – 24 hours)

Application of MALDI Mass-Spectrometry for Diagnostics of Particularly Dangerous Infectious Diseases: Current State of Affairs and Prospects

Mass spectrometry is a modern physical-chemical analytical method that provides for qualitative and quantitative assessment of the substance composition. It is based on pre-ionization of the atoms and molecules included into it. One of the advanced methods of ionization, due to which mass-spectrometry investigation of macromolecules has become a frequent practice, is matrix-assisted laser desorption/ionization (MALDI). The essence of it is the pulsed laser irradiation of the matter under study, mixed with the matrix. The review discusses current data on MALDI mass-spectrometry application for the performance of species-specific and genus-specific identification of microorganisms at the premises of diagnostic laboratories. Considered are the basic advantages of MALDI-TOF identification as compared to bacteriologic, immunologic, and molecular-genetic methods of assessment. Allocated is the mass-spectrometry position in the system of laboratory diagnostics of infectious diseases, including particularly dangerous ones, in the territory of the Russian Federation

Non-Specific Indication of Microorganisms in Environmental Samples

The review presents an analysis of modern methods and instruments for performing nonspecific detection of pathogenic biological agents in environmental objects. Discussed are technological characteristics of application of these methods for the detection of biological substances of protein nature in samples. The spectrum of means for non-specific PBA detection includes home-produced and foreign field devices based on protein contamination indication using various colorimetry variants. Technologies for remote and direct monitoring of environment for the presence of aerosols of biological nature are represented by hybrid lidar systems (biolidars) and biodetectors. For PBA nucleic acids tracing, the complexes based on DNA molecule binding with fluorophore with further fluorescence detection are described. Given are the examples of chemiluminescent analysis application in the developed automatic impurity detectors, as well as systems using bioluminescence. Based on the literature data analysis, put forward is a possible algorithm for indication of pathogenic biological agents when carrying out monitoring of the environment in zones of possible emergency situation occurrence and mass events holding

Long-Term Persistence of <i>Yersinia pestis</i> in Association with Acanthamoeba castellanii in Experiment

The aim of the study was to test the feasibility of long-term survival and preservation of the properties of Yersinia pestis in association with soil amoeba Acanthamoeba castellanii. Materials and methods. Y. pestis strains and acanthamoeba isolated in the common area of the Gorno-Altai high-mountain plague focus were used for the study. The systematic affiliation of protozoa was determined through analyzing the 18S rRNA gene fragment sequencing data, followed by alignment with amoeba sequences from the NCBI GenBank database. A fluorescent Y. pestis strain was obtained by electroporation using the pTurboGFP-B plasmid. Co-cultivation was carried out in saline buffer in the absence of nutrients for the cells of plague pathogen. The influence of co-culturing with protozoa on Y. pestis properties was determined using microbiological, biological, and molecular-genetic methods. Results and discussion. The cell viability preservation for 22 months of the experiment in Y. pestis strain belonging to the main subspecies of the antique biovar, the 4.ANT phylogenetic line in co-culture with amoeba cells in the absence of additional nutrients has been established. Co-cultivation with amoebae did not lead to a change in the cultural, morphological, genetic and virulent properties of the plague pathogen strain. The data obtained confirm the possibility of using Acanthamoeba castellanii by the plague microbe to persist in soil biocenoses and open up the prospect of studying the mechanisms of plague pathogen surviving during extended inter-epizootic periods

Anesthesia of laboratory animals in manufacturing of diagnostic and preventive biomedicines

Preparations such as XilaVet, Zoletil 100 as well as Aeranne (Isoflurane) are successfully applied for animal anesthesia in veterinary practice. We assessed a possibility of using parenteral narcosis with Zoletil 100 in combination with muscle relaxant Xila for producer-rabbits involved in manufacturing of natural rabbit serum subsequently deployed for production of diagnostic serum and immunoglobulin preparations. Administration of preparations into auricular vein is easy to do, while animals are sedated immediately allowing for safe fixation on restraining table and causing no additional stress for biomodels. This type of narcosis provides for expected depth of anesthesia and its maintenance until the end of blood-letting procedure. Parameters characterizing the state of cardiovascular system due to anesthetic products remained within the permitted limits. These preparations do not reduce heart beat rate allowing for collecting sufficient blood volumes. Application of inhalation anesthesia with Aeranne in laboratory animals provides for the specified depth of anesthesia and its maintenance until the end of the whole procedure. However, it requires specialized equipment and highly trained personnel with appropriate skills. Usage of Xila as a mono narcosis is not recommended as exhibits weak analgesic effects and strong hypotensive activity by decreasing quantity of collected blood volume. It was found that anesthetics such as Xila, Zoletil 100, Aeranne did not affect specific activity of immune sera in case of total dehematizing procedure. Moreover, antibody titers were not declined throughout entire observation (12 months) period and complied with the requirements of regulatory documentation. In addition, a feasibility of replacing old-fashioned anesthesia method with diethyl ether for a combination of safer contemporary preparations of Zoletil 100 and Xila was demonstrated while manufacturing tableted chemical cholera vaccine in experimental series with suckling rabbits used at diverse stages of raw material verification during surgical interventions. Xila, Zoletil 100, and Aeranne examined by us had no impact on the amount of blood obtained from donor-animals, immunological properties of the sera and ready-to-use diagnostic preparations. Such drugs were safe for all-age animals that comply with the requirements to anesthesia of animal biomodels and producer-animals in manufacturing of immunobiological preparations. Thus, our study allowed to conduct experiments with laboratory animals in a more humane manner

MALDI-TOF Mass-Spectrometry Analysis of Plague Agent Strains

Objective of the study was to demonstrate practicability of data base creation, containing reference mass-spectra of agents of particularly dangerous infections, using MALDI-TOF mass-spectrometry, by the example of plague agent strains. Materials and methods. MALDI-TOF mass-spectrometry was deployed for the obtainment of mass-spectra of ribosomal proteins from the microorganisms under investigation with the help of mass-spectrometers - Microflex LT. Results and conclusions. Carried out was comparative analysis of the obtained mass-spectra of 10 Y. pestis strains and reference spectra of Y. tuberculosis , contained in commercial data base of MALDI Biotyper 3.1 (Bruker Daltonics, Germany). Developed data base was validated in the process of identification of plague microbe strains, isolated in the territory of natural plague foci of the Russian Federation. That data base provided for correct identification of Y. pestis strains up to a species

Effectiveness of the “Sterius 60” SHF Radiation Installation for Disinfection of Objects Contaminated with PBA of Groups I–IV, when Working with Infected Biomodels

The aim was to evaluate the effectiveness of using the “Sterius 60” microwave disinfection system (Russia) for decontamination of objects infected with PBA of groups I–IV emerging as a result of working with infected laboratory animals.Materials and methods. Effectiveness verification of disinfection of biological waste generated as a result of the life of laboratory animals by SHF radiation was carried out in the microwave system “Sterius 60”, recommended by the manufacturer for disinfection of epidemiologically hazardous and extremely dangerous medical waste, including biological ones (classes B and C), by volumetric SHF heating. Carcasses of uninfected laboratory animals (white mice, Guinea pigs, suckling rabbits), granulated feed and bedding material (wood shavings), which are objects directly in contact with biomodels, were used as vivarium waste to be decontaminated. The following microorganisms were utilized as model test ones: Bacillus subtilus VKM B-911, Bacillus stearothermophilus VKM B-718, Bacillus licheniformis G VKM B-1711-D, Alcaligenes faecalis 415, Yersinia pestis EV, Bacillus anthracis STI. Laboratory utensils (plastic Petri dishes, porcelain mortars and pestles) were used as a mock-up chamber filler for model test microorganisms.Results and discussion. As a result of the study, data were obtained indicating that the microwave system for disinfection of medical waste “Sterius 60” is ineffective for decontamination of biological waste in laboratories working with biomodels infected with PBA of groups I–II. The established standard mode of disinfection of this system was effective only for non-spore forms of microorganisms, pathogenicity groups III–IV. Therefore, in our opinion, it is advisable to use it for decontamination of laboratory utensils infected with PBA of groups III–IV, directly at sites of waste generation

Specific Appurtenance, Numbers, and Dynamics of Interaction of Acanthamoeba from Soils of Gorno-Altai High-Mountain Plague Focus with Yersinia pestis Strains

Objective of the study is to analyze the appurtenance, numbers, and dynamics of interaction of acanthamoeba from soils of GornoAltai plague focus with Yersinia pestis 367 strain, isolated in 2016 in enzootic territory of this focus. Materials and methods. Utilized were soil amoeba from Gorno-Altai high-mountain focus and the strain Y. pestis 367 of the main subspecies of antique biovar, isolated there in 2016. Determination of systematic relation of the isolated amoeba was carried out using PCR with genus specific primers and sequencing of the obtained PCR fragments followed by identification of nucleotide sequences against GenBank database. Localization of Y. pestis cells in acanthamoeba was performed using fluorescent antibody technique by means of Axio Imager Z2 (Carl Zeiss, Germany). Results and conclusions. For the first time ever established has been the presence of Acanthamoeba castellanii in soils of burrows of Marmota altaica in the numbers of up to 300000 cells/gr in Gorno-Altai high-mountain focus. Investigated has been the dynamics of interaction of these microorganisms. Preservation of the agent in vacuoles of endoplasmatic reticulum within 14 days has been revealed. It is an indicative of the possibility of Y. pestis persistence in amoeba of Acanthamoeba subspecies in soil biocoenosis of Gorno-Altai high-mountain plague focus