The possibility of identifying individual strains of glanders and melioidosis pathogens with a combination of immunoblotting and DNA amplification methods

Causative agents of melioidosis and glanders are among the most dangerous bacterial pathogens for human. Moreover, Burkholderia pseudomallei and Burkholderia mallei are considered to be potential bioterrorism agents. In connection with this, timely diagnostics of such bacteria is of high importance. In our study, we made an attempt to develop an approach for detecting pathogenic Burkholderia spp. by combining species-specific amplification and strain-specific dot blotting assay with monoclonal antibodies. The following pathogenic Burkholderia strains were used in experiments: B. mallei (C-4, C-5, t-12, B-120, P-1, Мuksuwar-11, Z-12,Zagreb, Ivanovich, 5534), and B. pseudomallei (100, 102, 115, 116, 132, 135, 301, 51274, 60913, 61503). Real-Time PCR (RT-PCR) and dot blotting with monoclonal antibodies against surface Burkholderia epitopes were used to detect such pathogens. RT-PCR was carried out by using primers designed to recognize DNA fragments in B. mallei IS407A-fliP and the gene Orf12 from B. pseudomallei. For this, DNA was isolated from bacterial cells suspended at 1 × 104 microbial cells/ml. accumulation of the end reaction products was visualized by staining with dye SYBR Green I. Specificity of amplification reaction was determined by measuring melting temperature (Tm) for end products followed by running gel electrophoresis. It was demonstrated that all ten strains of either B. mallei or B. pseudomallei examined in the study were detected by using primers against IS407A-fliP DNA fragment and the gene Orf12, respectively. It was demonstrated that all ten strains of either B. mallei or B. pseudomallei examined in the study were detected by using primers against IS407A-fliP DNA fragment and the gene Orf12, respectively. Importantly, no signals specific to heterologous microbial DNA (isolated from bacterial cell suspension at concentration of 1 × 107 microbial cells/ml) were detected by using RT-PCR. Thus, RT-PCR provides an opportunity for assessing an inter-species diversity among pathogenic Burkholderia species. A genus-specificity was observed by using monoclonal antibodies 3D3 which bind to both Burkholderia strains, whereas antibodies 2D11 exhibited no selective binding to strain Р1 B. mallei and strain 100 B. pseudomallei, thereby displaying a strain-specific interaction. Thus, it allowed to conclude that combining a species-specific DNA amplification particularly RT-PCR together with immune-based assay such as dot blotting by using a panel of monoclonal antibodies seems to be a promising approach for assessing intra-species diversity among pathogenic Burkholderia

Application of immunomagnetic separation for accelerated detection of <i>F. tularensis</i> cells in soil samples using an immunochromatographic test

Introduction. Epizootological monitoring of the area contamination with the causative agent of tularemia implies the collection and analysis of a variety of field specimens. The analysis of such objects is time- and labour-consuming. In this context, simple and fast diagnostic techniques are needed to analyze specimens under resource-limited conditions. Aim. To study the possibility of using immunomagnetic separation for accelerated detection of Francisella tularensis cells in soil samples using immunochromatography. Materials and methods. Immunomagnetic particles (IMPs) were produced by using monoclonal antibodies to lipopolysaccharide (LPS) of the tularemia causative agent. Soil specimens weighing 1 g with preliminary introduced inactivated F. tularensis 15/10 cells were used in the study. The samples were suspended in an extraction buffer (EB) and filtered. Tularemia cells were separated by IMP suspension. The particles were washed, resuspended in EB and heated at 100C for 5 minutes. The supernatant was analyzed with test strips based on F. tularensis IC-test kit. Results. A combination of the immunomagnetic separation method and the IC test to detect F. tularensis cells identified up to 1 106 cells of the tularemia pathogen in analyzed soil samples, while 1 107 cells were detected in soil washouts in the absence of immunomagnetic separation. Conclusion. The developed technique combining immunomagnetic separation and IC tests opens up prospects for express diagnostics of soil sample contamination in tularemia foci. The analysis takes about 3 hours, and its sensitivity is 1 106 cells/g of soil. The technique is simple, not requiring sophisticated expensive equipment. It can be easily adapted for testing other specimen types (water, grain, etc.). In addition, separated bacterial cells can be used for F. tularensis detection by other methods

The role of resuscitation promoting factors in pathogenesis and reactivation of Mycobacterium tuberculosis during intra-peritoneal infection in mice

<p>Abstract</p> <p>Background</p> <p><it>Mycobacterium tuberculosis </it>can enter into a dormant state which has resulted in one third of the world's population being infected with latent tuberculosis making the study of latency and reactivation of utmost importance. <it>M. tuberculosis </it>encodes five resuscitation promoting factors (Rpfs) that bear strong similarity to a lysozyme-like enzyme previously implicated in reactivation of dormant bacteria <it>in vitro</it>.</p> <p>We have developed an intraperitoneal infection model in mice, with immune modulation, that models chronic infection with similar properties in mouse lungs as those observed in the murine aerosol infection model. We have assessed the behavior of mutants that lack two or three <it>rpf </it>genes in different combinations in our intraperitoneal model.</p> <p>Methods</p> <p>C57Bl/6 mice were intraperitonealy infected with H37Rv wild type <it>M. tuberculosis </it>or mutant strains that lacked two or three <it>rpf </it>genes in different combinations. After 90 days of infection aminoguanidine (AG) or anti-TNFα antibodies were administrated. Organ bacillary loads were determined at various intervals post infection by plating serial dilutions of organ homogenates and enumerating bacteria.</p> <p>Results</p> <p>We found that the <it>rpf </it>triple and double mutants tested were attenuated in their ability to disseminate to mouse lungs after intraperitoneal administration and were defective in their ability to re-grow after immunosuppression induced by administration of aminoguanidine and anti-TNFα antibodies.</p> <p>Conclusion</p> <p>Rpf proteins may have a significant physiological role for development of chronic TB infection and its reactivation <it>in vivo</it>.</p

Identification of T-Cell Antigens Specific for Latent Mycobacterium Tuberculosis Infection

BACKGROUND: T-cell responses against dormancy-, resuscitation-, and reactivation-associated antigens of Mycobacterium tuberculosis are candidate biomarkers of latent infection in humans. METHODOLOGY/PRINCIPAL FINDINGS: We established an assay based on two rounds of in vitro restimulation and intracellular cytokine analysis that detects T-cell responses to antigens expressed during latent M. tuberculosis infection. Comparison between active pulmonary tuberculosis (TB) patients and healthy latently M. tuberculosis-infected donors (LTBI) revealed significantly higher T-cell responses against 7 of 35 tested M. tuberculosis latency-associated antigens in LTBI. Notably, T cells specific for Rv3407 were exclusively detected in LTBI but not in TB patients. The T-cell IFNgamma response against Rv3407 in individual donors was the most influential factor in discrimination analysis that classified TB patients and LTBI with 83% accuracy using cross-validation. Rv3407 peptide pool stimulations revealed distinct candidate epitopes in four LTBI. CONCLUSIONS: Our findings further support the hypothesis that the latency-associated antigens can be exploited as biomarkers for LTBI

Proteins of the Rpf Family: Immune Cell Reactivity and Vaccination Efficacy against Tuberculosis in Mice

It was shown recently that Mycobacterium tuberculosis expresses five proteins that are homologous to Rpf (resuscitation promoting factor), which is secreted by growing cells of Micrococcus luteus. Rpf is required to resuscitate the growth of dormant Micrococcus luteus organisms, and its homologues may be involved in mycobacterial reactivation. Mycobacterial Rpf-like products are secreted proteins, which makes them candidates for recognition by the host immune system and anti-Rpf immune responses potentially protective against reactivated tuberculosis. Here we report that the Rpf protein itself and four out of five of its mycobacterial homologues, which were administered as subunit vaccines to C57BL/6 mice, are highly immunogenic. Rpf-like proteins elicit immunoglobulin G1 (IgG1) and IgG2a responses and T-cell proliferation and stimulate production of gamma interferon, interleukin-10 (IL-10), and IL-12 but not IL-4 or IL-5. Both humoral and T-cell responses against these antigens show a high degree of cross-reactivity. Vaccination of mice with Rpf-like proteins results in a significant level of protection against a subsequent high-dose challenge with virulent M. tuberculosis H37Rv, both in terms of survival times and mycobacterial multiplication in lungs and spleens