The facultative intracellular pathogen Francisella tularensis is the causative agent of the serious infectious disease tularemia. Despite intensive research, the virulence factors and pathogenetic mechanisms remain largely unknown. To identify novel putative virulence factors, we carried out a comparative proteome analysis of fractions enriched for membrane-associated proteins isolated from the highly virulent subspecies tularensis strain SCHU S4 and three representatives of subspecies holarctica of different virulence including the live vaccine strain. We identified six proteins uniquely expressed and four proteins expressed at significantly higher levels by SCHU S4 compared to the ssp. holarctica strains. Four other protein spots represented mass and charge variants and seven spots were charge variants of proteins occurring in the ssp. holarctica strains. The genes encoding proteins of particular interest were examined by sequencing in order to confirm and explain the findings of the proteome analysis. Our studies suggest that the subspecies tularensis-specific proteins represent novel potential virulence factors.
The facultative intracellular bacterium Francisella tularensis is the causal agent of the serious infectious disease tularemia. Despite the dynamic progress, which has been made in last few years, important questions regarding Francisella pathogenicity still remain to be answered. Generally, secreted proteins play an important role in pathogenicity of intracellular microbes. In this study, we investigated the protein composition of the culture filtrate proteins of highly virulent F. tularensis subsp. tularensis, strain SCHU S4 and attenuated F. tularensis subsp. holarctica, live vaccine strain using a comparative proteomic analysis. The majority of proteins identified in this study have been implicated in virulence mechanisms of other pathogens, and several have been categorized as having moonlighting properties; those that have more than one unrelated function. This profiling study of secreted proteins resulted in the unique detection of acid phosphatase (precursor) A (AcpA), β-lactamase, and hypothetical protein FTT0484 in the highly virulent strain SCHU S4 secretome. The release of AcpA may be of importance for F. tularensis subsp. tularensis virulence due to the recently described AcpA role in the F. tularensis escape from phagosomes.
Immunoproteomic analysis was applied to study the immunoreactivity of serum samples collected at different time points from a laboratory assistant accidentally infected with highly virulent strain of Francisella tularensis subsp. tularensis. Immunoblotting showed that the spectrum of F. tularensis antigens recognized specifically by immune sera remained with the exception for 1 antigen stable for up to 16 years after infection. Using immunoproteomics approach 10 immunoreactive antigens were successfully identified. Several new immunogenic F. tularensis proteins were described for the first time.
ABSTRACT:We tested the germicide activity of 1% Chloramin BM, 1% Incidin Plus, 1% Lysoformin 3000, 0.2% Mikasept KP, and 2% Sekusept Forte against viruses in suspension (suspension test) and dried onto a surface (carrier test). The agents of the porcine encephalomyelitis (Porcine teschovirus, strains CAPM V-86, CAPM V-37), Aujeszky's disease (strains CAPM V-166, CAPM V-327) and vesicular stomatitis (strains CAPM V-499, CAPM V-331) were used as model viruses. After 30 min contact time in both the suspension and carrier tests, the Porcine teschovirus was 4 lg inactivated only by Mikasept, which was thus the only disinfectant to meet the standard. The other disinfectants decreased the viral titre insufficiently. Under the same conditions, Aujeszky's disease virus was inactivated by at least 4 lg by all the tested disinfectants except for Chloramin BM which decreased the titre of CAPM V-166 only by 3.75 lg in the carrier test. For the inactivation of Vesicular stomatitis virus Chloramin BM and Mikasept KP were tested. Both the disinfectants reliably decreased the viral titre in both the suspension and carrier tests. Our results show that the inactivation of a surface-bound virus is more difficult than its inactivation in suspension. We confirm the high resistance of non-enveloped viruses (Porcine teschovirus) to chemical inactivation.
Contamination of cell cultures and sera used for animal virus propagation with mycoplasmas represents a serious problem, especially in virology. Therefore specific control measures must be used. To achieve this we introduced PCR for the detection of mycoplasma species in cell cultures and compared its results with ELISA and microbiological culture. Seven mycoplasma species which are the most common contaminants of cell lines (Mycoplasma arginini, M. fermentans, M. hyorhinis, M. bovis, M. orale, M. hominis, and Acholeplasma laidlawii) were used to verify the method. Then we assessed five selected cell lines and three bovine sera by the PCR, ELISA and culture methods and compared the results. PCR was positive for all of the mycoplasma species tested. ELISA kit used (Mycoplasma detection kit, Roche, Germany) allowed detection of only four species of contaminating mycoplasmas (Acholeplasma laidlawii, Mycoplasma arginini, M. hyorhinis, and M. orale). All the methods detected contamination of the VERO and RK13 cell lines. The agents of contamination were determined by the species-specific ELISA kit as Mycoplasma arginini and M. orale, respectively. Other cell lines and sera tested were not contaminated with mycoplasma. The results confirmed that the PCR method used in the present study is a sensitive, fast and specific detection method of mycoplasma contaminations and is suitable for routine mycoplasma detection in cell cultures and bovine sera.
Early detection of biohazardous bacteria that can be misused as biological weapons is one of the most important measures to prevent the spread and outbreak of biological warfare. For this reason, many instrument platforms need to be introduced into operation in the field of biological warfare detection. Therefore the purpose of this study is to establish a new detection panel for biothreat bacteria (Bacillus anthracis, Yersinia pestis, Francisella tularensis, and Brucella spp.) and confirm it by collaborative validation by using a multiplex oligonucleotide ligation followed by polymerase chain reaction and hybridization to microspheres by MagPix detection platform (MOL-PCR). Appropriate specific sequences in bacterial DNA were selected and tested to assemble the detection panel, and MOLigo probes (short specific oligonucleotides) were designed to show no cross-reactivity when tested between bacteria and to decrease the background signal measurement on the MagPix platform. During testing, sensitivity was assessed for all target bacteria using serially diluted DNA and was determined to be at least 0.5 ng/µL. For use as a diagnostic kit and easier handling, the storage stability of ligation premixes (MOLigo probe mixes) was tested. This highly multiplex method can be used for rapid screening to prevent outbreaks arising from the use of bacterial strains for bioterrorism, because time of analysis take under 4 h.
ABSTRACT:We studied the effectivity of a commercial antibiotic kit Mycokill AB for the elimination of mycoplasma contamination in virus strains. The contaminated virus strains were first filtered, treated with Mycokill AB for three hours and repeatedly passaged in its presence in the cultivation medium in pure cell lines. Three passages in the presence of Mycokill AB were invariably followed by three passages without Mycokill AB. The effectivity of purification was then checked by PCR. Twenty-four out of 28 tested virus strains became free of mycoplasma after a first or second cycle of the treatment with Mycokill AB. The other four strains remained positive even after repeated passages. In these cases of a likely resistance to Mycokill AB, we managed to eliminate the mycoplasma contamination through a subsequent treatment with the antibiotic combination BM-Cyclin. Mycokill AB was shown in the elimination of mycoplasma from virus suspensions as successful as other known most effective antibiotics.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.