Extraction and Serological Properties of Mycobacterium Cell Surface and Excreted Proteins

© 2017, Springer Science+Business Media, LLC, part of Springer Nature. Modern medicine still faces the task of distinguishing active and latent tuberculosis cases at the early stage of the disease. Serological approaches have their advantages for their use in diagnostics. However, the progress of these approaches is ongoing but further progress is needed to meet the needs for this disease. Here, we extracted Mycobacterium tuberculosis H37Rv proteins from culture medium or from the cell surface and studied their reactivity with anti-M. tuberculosis serum in both ELISA and immunoblots. We found that M. tuberculosis surface proteins, extracted using dimethyl sulfoxide, with molecular weights in the range of 6.5–200 kDa, showed strong specific reactivity with anti-M. tuberculosis positive serum. While excreted proteins in the molecular weight range of 32–45 kDa had the highest reactivity. The latter was confirmed serologically when very weak signal was detected from the filtrate fractions compared to stronger activity from the Vivaspin 50 kDa MWCO retentates. Moreover, Mycobacterium bovis and tuberculosis proteins from the filter retentates had clear specific serum reactivity, which suggests that this approach can be used for differential diagnosis of two infections

Novel M. tuberculosis specific IL-2 ELISpot assay discriminates adult patients with active or latent tuberculosis

© 2018 Della Bella et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Background Tuberculosis (TB) still is a major worldwide health problem, with 10.4 million new cases in 2016. Only 5–15% of people infected with M. tuberculosis develop TB disease while others remain latently infected (LTBI) during their lifetime. Thus, the absence of tests able to distinguish between latent infection and active tuberculosis is one of the major limits of currently available diagnostic tools. Methods A total of 215 patients were included in the study as active TB cases (n = 73), LTBI subjects (n = 88) and healthy persons (n = 54). Peripheral blood mononuclear cells (PBMCs) were isolated from each patient and the LIOSpot® TB anti-human IL-2 ELISpot assay was performed to test their proliferative response to M. tuberculosis antigens ESAT-6, CFP-10 and Ala-DH. Statistical analysis was performed to define the sensitivity and the specificity of the LIOSpot® TB kit for each antigen used and to set the best cut off value that enables discrimination between subjects with active TB or latent TB infection. Results Comparing the LIOSpot® TB results for each tested antigen between uninfected and infected subjects and between people with latent infection and active TB disease, the differences were significant for each antigen (p< 0.0001) but the ROC analysis demonstrated a high accuracy for the Ala-DH test only, with a cut off value of 12.5 SFC per million PBMCs and the Ala-DH ROC curve conferred a 96% sensitivity and 100% specificity to the test. For the ESAT-6 antigen, with a best cut off value of 71.25 SFC per million PBMCs, a sensitivity of 86% and specificity of 36% was obtained. Finally, the best cut off value for CFP-10 was 231.25 SFC per million PBMCs, with a sensitivity of 80% and a specificity of 54%. Thus, as for IGRA assays such as Quantiferon and T-Spot TB tests, ESAT-6 and CFP-10 are unable to distinguish LTBI from active TB when IL-2 is measured. On the contrary, the IL-2 production induced by Ala-DH, measured by LIOSpot® TB kit, shows high sensitivity and specificity for active TB disease. Conclusions This study demonstrates that the LIOSpot® TB test is a highly useful diagnostic tool to discriminate between latent TB infection and active tuberculosis in adults patients

Seasonal variation in heavy-metal accumulation in honey bees as an indicator of environmental pollution

Determination of contaminants in biological objects is one of the most important aspects of environmental health control. The objective of this study was to investigate heavy metals accumulation in bees at different seasons in areas with different degrees of technogenic pollution. The concentrations of six microelements in 36 honey bee samples taken from uncontaminated and contaminated environments were analyzed by atom-absorption spectrometry. The contents of heavy metals in bee samples differed significantly by season and location. Cadmium and manganese were found in higher concentrations in summer bees from contaminated apiaries than in autumn ones. On average, levels of cadmium, lead, manganese in contaminated areas exceeded those in control areas by 3.3, 4.5, 2.3 times respectively. There was no discrepancy found in concentration of heavy metals between summer and autumn bees from control apiaries. The index for evaluation of degree of technogenic contamination with used concentrations in different seasonal generations was proposed (ratio of heavy metal concentration of summer and autumn bee generations collected from the same colony). Therefore, honey bees can be used to evaluate contamination in terrestrial ecosystems

Efficiency of specific biopreparations in organic waste management

Background: Biological approach is becoming more popular in the protection of the environment by organic waste of agricultural holdings due to economic efficiency and absence of additional damage to the ecosystem. Microorganisms of biopreparations possess significant fermentative properties and high antagonistic activity against of many pathogenic and opportunistic bacteria and toxigenic filamentous fungi. In our study we have isolated millions of coliform bacteria and Enterococci, thousands of Salmonella, Proteus and Staphylococci in 1 g of initial substrate. After the usage of biopreparations the quantity of coliform bacteria and Enterococci was less than 10 cells/g, and no Salmonella, Staphylococcus or Proteus were found. Application of the biopreparations has prevented the loss of nutrients. Therefore, nitrogen content was higher by 50-60% than in the control. The quantity of nitrifying, ammonifying and cellulose-fermenting microorganisms increased by 15.9%, 6.6% and 15.4%, respectively. Productivity of grain and vegetable crops increased by 10-20%. An important advantage of the biopreparations usage, that also we found, is the elimination of specific odor within a couple days due to the ability of their microorganisms to assimilate nitrogen from urea and neutralize the substrate against the bacteria causing putrefaction, anaerobic processes and the emission of ammonia and hydrogen sulphide. Conclusion: The application of developed new biopreparations will allow producing high-quality environmentally friendly agricultural products

Design of primers for identification of honey bee viruses in multiplex-PCR

This paper is devoted to the design of primer oligonucleotide sequences for their use in the genetic identification of Sacbrood virus, Chronic bee paralysis virus, Black queen cell virus and Deformed wing virus using multiplex-PCR. As a result of the bioinformatic analysis, the design of the oligonucleotide primers was performed; the designed primers had similar annealing temperatures (55 C), which makes it possible to indicate each of the viruses under the same PCR conditions. Most of the known strains and isolates of these viruses are amplified with this complex of oligonucleotide primers. Nucleotide sequences of designed primers and a universal positive control allow for the genetic identification of each of the biopathogens under the same PCR conditions at a multiplex format

Marker Loci in Brucella Genome for Differential PCR Indication of Pathogenic strains

Objective of this work was to develop the algorithms for differential PCR indication of Brucella genus strains using databases of their genomes. Materials and methods .Resources of the National Center for Biotechnology Information (NCBI) and BLAST and Vector NTI 9.1.0 software utilities. For PCR amplification, B. suis, B. abortus, B. melitensis nucleic acids, as well as plasmid DNA with marker insertions were used. Results and conclusions. We assessed brucella gene sequences, some of which are found in Brucella genus bacteria, others only in representatives of B. melitensis, and the third ones – only in representatives of B. abortus. As a result of primers and probes designing for indication of Brucella genus bacteria and representatives of B. melitensis and B. abortus species, criteria for marker sequence amplification have been established. These criteria provide for simultaneous differentiation in a single reaction. The determination of strain differences within one species of Brucella is described in multilocus VNTR assay technique, and the profiles of tandem repeats of various B. melitensis and B. abortus strains are available in the public domain. To monitor the progress of amplification, a positive control has been developed that has the nucleotide sequence of all marker regions. The text of the paper discloses all the nucleotide sequences of primers, probes and positive control, which makes it possible to independently acquire them in competent organizations

Novel drug targets in cell wall biosynthesis exploited by gene disruption in Pseudomonas aeruginosa

© 2017 Elamin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. For clinicians, Pseudomonas aeruginosa is a nightmare pathogen that is one of the top three causes of opportunistic human infections. Therapy of P. aeruginosa infections is complicated due to its natural high intrinsic resistance to antibiotics. Active efflux and decreased uptake of drugs due to cell wall/membrane permeability appear to be important issues in the acquired antibiotic tolerance mechanisms. Bacterial cell wall biosynthesis enzymes have been shown to be essential for pathogenicity of Gram-negative bacteria. However, the role of these targets in virulence has not been identified in P. aeruginosa. Here, we report knockout (k.o) mutants of six cell wall biosynthesis targets (murA, PA4450; murD, PA4414; murF, PA4416; ppiB, PA1793; rmlA, PA5163; waaA, PA4988) in P. aeruginosa PAO1, and characterized these in order to find out whether these genes and their products contribute to pathogenicity and virulence of P. aeruginosa. Except waaA k.o, deletion of cell wall biosynthesis targets significantly reduced growth rate in minimal medium compared to the parent strain. The k.o mutants showed exciting changes in cell morphology and colonial architectures. Remarkably, ΔmurF cells became grossly enlarged. Moreover, the mutants were also attenuated in vivo in a mouse infection model except ΔmurF and ΔwaaA and proved to be more sensitive to macrophage-mediated killing than the wild-type strain. Interestingly, the deletion of the murA gene resulted in loss of virulence activity in mice, and the virulence was restored in a plant model by unknown mechanism. This study demonstrates that cell wall targets contribute significantly to intracellular survival, in vivo growth, and pathogenesis of P. aeruginosa. In conclusion, these findings establish a link between cell wall targets and virulence of P. aeruginosa and thus may lead to development of novel drugs for the treatment of P. aeruginosa infection

Preparation and use of transplantable cell line of newborn rabbits for reproduction of viruses

The purpose of the study was development of a way of receiving culture of cells from bodies of newborn rabbits for a reproduction of production strains of viruses

Isolation, Purification and Evaluation of Serological Activity of Rabies Virus Antigens

Objective of the study is to evaluate the serological activity of rabies virus antigens isolated from the brain tissue of mice by homogenization on FastPrep followed by ultracentrifugation. Materials and methods. Producer strain of the rabies virus “Ovechiy” GNKI. The rabies virus was isolated from the brain tissue of experimentally infected mice, followed by the study of the electrophoretic profile. The serological activity of the virus components was assessed by immunoblot and ELISA using specific anti-rabies sera.Results and conclusions. In the course of comparing the methods of isolation and purification of the rabies virus antigen, it was found that most optimal one is to use a homogenization on FastPrep-24, followed by fractionation in a sucrose gradient. As a result of fractionation in a graded sucrose density gradient with a concentration of 15–50 % at 25000 g for 120 min, five fractions of the rabies virus components were obtained. The maximum purified protein fraction was from 15–20 % sucrose zone, which corresponded to a molecular weight of 67 kDa. The specific antigen activity of the fraction in ELISA reached up the titers of 1:1280 (Specificity coefficient 2.2). Using immunoblot of antigens, obtained from the sucrose gradient in the range of 40–45 % and 20–35 % after ultracentrifugation, one major fraction of polypeptides (54 kDa) was detected, which showed the highest antigenic activity. The results obtained will be useful in the design of test systems for rabies screening and monitoring the effectiveness of anti-epizootic measures

Epidemiological dynamics of nephropathia epidemica in the Republic of Tatarstan, Russia, during the period of 1997-2013

Copyright © Cambridge University Press 2015.This report summarizes epidemiological data on nephropathia epidemica (NE) in the Republic of Tatarstan, Russia. NE cases identified in the period 1997-2013 were investigated in parallel with the hantavirus antigen prevalence in small rodents in the study area. A total of 13 930 NE cases were documented in all but one district of Tatarstan, with most cases located in the central and southeastern districts. The NE annual incidence rate exhibited a cyclical pattern, with the highest numbers of cases being registered once in every 3-5 years. The numbers of NE cases rose gradually from July to November, with the highest morbidity in adult males. The highest annual disease incidence rate, 64·4 cases/100 000 population, was observed in 1997, with a total of 2431 NE cases registered. NE cases were mostly associated with visiting forests and agricultural activities. The analysis revealed that the bank vole Myodes glareolus not only comprises the majority of the small rodent communities in the region, but also consistently displays the highest hantavirus prevalence compared to other small rodent species