Q fever and prevention.

Coxiella burnetii is an intracellular, Gram-negative bacterium and the etiological agent of Q fever, a worldwide zoonotic disease with a considerable economic impact in the livestock industry. Domesticated ruminants like cattle, sheep, and goats are the main reservoirs of Q fever in men. Humans usually acquire the disease by inhaling contaminated aerosol produced by infected livestock. Acute infection is typically asymptomatic or manifests as a febrile flu-like illness or pneumonia. In 1-5% of primary infections, chronic Q fever may develop, which can be life-threatening and often presents as endocarditis. Q fever outbreaks usually occur from occupational exposure involving shepherds, veterinarians, animal handlers, abattoir or dairy workers, and laboratory personnel working with C. burnetii. Thus, prevention and control efforts should be primarily directed toward these groups and the surrounding environment. The most effective way of prophylaxis is vaccination. Although clinical data are lacking, vaccination should also be considered for persons who are at higher risk for development of chronic Q fever, including those with cardiac valve defects, vascular aneurysms, and immunocompromised patients. Therefore, development of an effective and harmless vaccine is a subject of constant interest of researchers for decades. This review summarizes the progress toward Q fever vaccine development.

Counting of viable C. burnetii cells by quantitative reverse transcription PCR using a recombinant plasmid (pCB-dotA) as a standard.

Coxiella burnetii is an intracellular pathogenic bacterium and etiological agent of Q fever in humans. Recently, the bacterium has been set free from the strictly intracellular condition by successful cultivation in acidified citrate cysteine medium. Here, we report a bacterial cell counting method that allows rapid quantification of the absolute or relative number of live cells of C. burnetii in a high throughput manner. The method utilizes TaqMan-based quantitative polymerase chain reaction (qPCR) targeting a single dotA gene for determination of genome equivalent (GE) presented either as DNA or complementary DNA (cDNA) synthesized via reverse transcription. The assay was shown to be specific, sensitive and efficiently reproducible. The quantification was linear over a range of 30 to 3x108 copies. Since there is only one copy of the dotA gene per Coxiella chromosome, the calculated dotA copy numbers can be compared to the number of bacterial cells. Finally, we demonstrated the potential of the method to assess effects of antibiotic on cell viability and to determine the antibiotic-tolerant fraction within a cell population. Keywords: Coxiella burnetii; Q fever; real-time polymerase chain reaction; copy number; antibiotic; axenic media; dotA gene.

Workshop on Q fever.

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Protein composition of the phase I Coxiella burnetii soluble antigen prepared by extraction with trichloroacetic acid.

Q fever is a highly infectious, widespread airborne zoonosis caused by Coxiella burnetii bacterium. Humans usually acquire the disease by inhalation of contaminated aerosol produced by infected livestock. Vaccination is the most practical way for prevention and control of the disease in the exposed population. In this work, we reviewed the most important Q-fever outbreaks in Slovakia as well as the progress in vaccine development. One of them represents a soluble antigen complex produced by extraction with trichloroacetic acid from a highly purified C. burnetii phase I strain Nine Mile. It was developed at the Institute of Virology in Bratislava. The protein content of this vaccine was separated by gel electrophoresis and analyzed by mass spectrometry. The study has resulted in the identification of 39 bacterial proteins from which 12 were recognized as immunoreactive. Most of the proteins were involved in bacterium pathogenicity (41.6%) and cell wall maintenance (25%). Four of the immunoreactive proteins may possess the moonlighting activity. Definition of the vaccine components represents a prerequisite for vaccine standardization and approval by governmental authorities.

Two mice models for transferability of zoonotic bacteria via tick vector.

Spotted fever and typhus-related diseases caused by rickettsiae, Lyme borreliosis induced by spirochetes from Borrelia burgdorferii sensu lato complex, and Q fever evoked by Coxiella burnetii, are important zoonoses occurring worldwide. In order to study the pathogenesis of these infections, the efficacy of vaccines from the perspective of protection against the pathogens, pathogen - pathogen interactions during co-infections or pathogen-vector-host interrelationship, a suitable animal model should be established. In this study, we evaluated two mouse models - the C3H/N and Balb/c strains for susceptibility to infection and ability to transmit the pathogens via tick vector and to reveal the potential interactions between various bacterial tick-borne agents. Our results indicated that the C3H/N and Balb/c mice are well-accepted models of B. afzelii infection. However, they are not suitable for interaction studies with R. helvetica since the animals did not acquire rickettsiemia and do not transmit Rickettsia sp. to feeding ticks.

Coxiella burnetii immunogenic proteins as a basis for new Q fever diagnostic and vaccine development.

Coxiella burnetii is the etiological agent of the zoonosis Q fever, which can cause an acute or a chronic, life-threatening disease in humans. It presents a highly stable cell form, which persists in the environment and is transmitted via contaminated aerosols. Ruminants are considered as the main reservoir for human infections but are usually asymptomatic. Subclinical infection in these animals and the occurrence of serologically negative shedders hamper the identification of infected animals with the currently used diagnostic techniques. This suboptimal sensitivity limits reliable identification of infected animals as well as the well-timed implementation of countermeasures. This review summarizes compounds, focusing on C. burnetii seroreactive proteins, which were discovered in recent immunoproteomic studies. We analyzed these proteins regarding their localization, function, frequency of citation, differences seen in various host species as well as sensitivity and specificity. Finally, proteins useful for the development of new diagnostic test systems as well as subunit vaccines were discussed.

New way of purification of pathogenic rickettsiae reducing health risks.

In general, cultivation and purification of intracellular pathogenic rickettsiae represents a risk for laboratory personnel due to exposure to highly infectious aerosol or accidental inoculation during these procedures. In this study, we describe an alternative, effective and time saving technique for rickettsial purification using digitonin to release intracellular bacteria from host cell without physical disruption. No significant differences were noted in yield and infectivity between digitonin treated rickettsiae and rickettsiae purified by sonication. This is the first report of using digitonin in purification of pathogenic rickettsiae and this approach might be effective for other intracellular pathogenic bacteria.

Modifications in the glycerophospholipid composition between the Coxiella burnetii phase I and phase II cells suggest an association with phase variation of the bacterium.

Glycerophospholipids (GP) extracted from the Coxiella burnetii strain Nine Mile in virulent phase I (NM I) and low virulent phase II (NM II) were analyzed by Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) that gave a superior mass resolution and mass accuracy allowing unambiguous peak recognition and precise assignment of ions. We showed that GP present in the pathogen's outer membrane underwent considerable modifications during the phase variation that might be related to impact of various environmental factors. It was found that GP from phase I cells were much more complex than those from phase II cells. While glycerophosphoethanolamines (PE), glycerophosphocholines (PC) and glycerophosphoglycerols (PG) were present in both phases of C. burnetii, major differences were observed in the presence of glycerophosphates (PA) and glycerophosphoserines (PS). Thus, PA but no PS were detected in NM I variant in contrast with NM II cells where PS but no PA were identified. It is suggested that enzymes for PA head group modifications to form PS, PE, and PG become active during the phase variation of the bacterium.

Salt-induced subcellular kinase relocation and seedling susceptibility caused by overexpression of Medicago SIMKK in Arabidopsis.

Dual-specificity mitogen-activated protein kinases kinases (MAPKKs) are the immediate upstream activators of MAPKs. They simultaneously phosphorylate the TXY motif within the activation loop of MAPKs, allowing them to interact with and regulate multiple substrates. Often, the activation of MAPKs triggers their nuclear translocation. However, the spatiotemporal dynamics and the physiological consequences of the activation of MAPKs, particularly in plants, are still poorly understood. Here, we studied the activation and localization of the Medicago sativa stress-induced MAPKK (SIMKK)-SIMK module after salt stress. In the inactive state, SIMKK and SIMK co-localized in the cytoplasm and in the nucleus. Upon salt stress, however, a substantial part of the nuclear pool of both SIMKK and SIMK relocated to cytoplasmic compartments. The course of nucleocytoplasmic shuttling of SIMK correlated temporally with the dual phosphorylation of the pTEpY motif. SIMKK function was further studied in Arabidopsis plants overexpressing SIMKK-yellow fluorescent protein (YFP) fusions. SIMKK-YFP plants showed enhanced activation of Arabidopsis MPK3 and MPK6 kinases upon salt treatment and exhibited high sensitivity against salt stress at the seedling stage, although they were salt insensitive during seed germination. Proteomic analysis of SIMKK-YFP overexpressors indicated the differential regulation of proteins directly or indirectly involved in salt stress responses. These proteins included catalase, peroxiredoxin, glutathione S-transferase, nucleoside diphosphate kinase 1, endoplasmic reticulum luminal-binding protein 2, and finally plasma membrane aquaporins. In conclusion, Arabidopsis seedlings overexpressing SIMKK-YFP exhibited higher salt sensitivity consistent with their proteome composition and with the presumptive MPK3/MPK6 hijacking of the salt response pathway.

Recent progress in glycomics and proteomics of the Q fever bacterium Coxiella burnetii.

Coxiella burnetii is an intracellular, Gram-negative bacterium and causative agent of Q fever. In humans, the disease ranges mostly from a flu-like illness and self-recovering mild pneumonia to severe meningoencephalitis, myocarditis or endocarditis. Recent molecular and biochemical/immunological advances, along with improved instrumentation, have provided unique insight into the host-parasite interrelationship and revealed previously unknown virulence strategies of C. burnetii. Noticeable progress has also been achieved in gaining a better understanding of the role of two major outer membrane components - lipopolysaccharide (LPS) and proteins in the life and immunopathobiology of the bacterium. Detailed glycomic studies have brought indispensable structural and functional information on LPS and its role in pathogenesis and immunity of Q fever. Recent proteomic studies have brought a deeper insight into the pathogen`s physiology, virulence and development and offered new possibilities in the investigation of inter/intra-species variation. This review will focus on advances in glycomics and proteomics of C. burnetii providing information on unique glycan and protein species, which together with other findings in the field, might lead to both a better understanding of this unusual pathogen and improvements in Q fever diagnosis and therapy.

Some possibilities of an analysis of complex samples by a mass spectrometry with a sample pretreatment by an offline coupled preparative capillary isotachophoresis.

This work deals with an analysis of biologically important compounds in complex matrices using preparative isotachophoresis (pITP) in column coupling configuration as a sample pretreatment technique followed by a direct infusion mass spectrometry with nano-electrospray ionization (DI-nESI-MS). Busereline was chosen as a model analyte, and urine was chosen as an example of complex matrix. In pITP experiments, sodium cation (10 mmol/L concentration) was used as a leading ion and ß-alanine as terminating ion (20 mmol/L concentration). The fractions, obtained by pITP pre-separation with the assistance of the mixture of discrete spacers, were finally analyzed by DI-nESI-MS. It was shown that pITP performed before DI-nESI-MS analysis can significantly simplify complex matrix, and, due to its concentration power, pITP can consequently decrease the concentration limit of detection. The concentration of buserelin in the urine samples analyzed by pITP-DI-nESI-MS was 10 µg/L (reflecting at a 8.10⁻9 mol/L concentration) in our work but from the ion intensities obtained in MS as well as MS/MS analyses, it is clear that this concentration level could be several orders of magnitude lower for reliable detection and identification of buserelin in urine analyzed using pITP with DI-nESI-MS detection.

Characterization of antigens for Q fever serodiagnostics.

The aim of this study was to identify candidate proteins for serodiagnostics of Q fever by monoclonal antibodies (MAbs), and to clone, express, and purify the selected proteins for use as antigens in ELISA. The reactivity of three MAbs to Coxiella burnetii (C. b.) Nine Mile strain and one MAb to Priscilla strain was tested using SDS-PAGE, 2-D gel electrophoresis, immunoblot analysis, and mass spectrometry. Three immunoreactive Q fever-specific proteins discriminated by MAbs, namely the CBU_0937 protein, outer membrane Com1 (CBU_1910) protein, and elongation factor Tu (CBU_0236) were identified. Successful PCR-amplification, cloning, expression, and purification of the recombinant proteins Com1 and CBU_0937 allowed their use for the screening of sera from patients with Q fever endocarditis (18) or acute Q fever (16) in ELISA. The recombinant protein CBU_0937 with unknown biological function proved to be a more applicable diagnostic tool for Q fever ELISA as compared to the Com1 protein.

Presence of serum carbonic anhydrase autoantibodies in patients relapsed after autologous stem cell transplantation indicates an improved prognosis.

Here we report patients with Hodgkin's disease and multiple myeloma, who relapsed/progressed after high dose therapy and autologous stem cell transplantation. In patients who developed aplastic anemia type syndrome, spontaneous tumor regression was observed and concomitantly high titers of serum autoantibodies were found. In order to identify the antibody specificity, two-dimensional electrophoresis, blotting and immunoreactions were used to analyze the peripheral blood stem cell extract with autoantibodies containing serum. The unique protein spot visualized exclusively by serum of patients with aplastic anemia type syndrome was identified as carbonic anhydrase I (CA I, accession No. P00915 and Q7M316) by means of mass spectrometry. The specificity of autoantibodies was confirmed by reaction with commercial CAs I, II, IX and XII. Immunoreaction in Western blots with these CA isoforms differed in sera obtained from patients with various types of the disease. Sera of Hodgkin's disease patients reacted with CA I, II and XII; sera of multiple myeloma patients reacted with the CA I, II, XII and IX. Patients developing and/or possessing CA autoantibodies had a significant survival benefit over those who did not develop CA anhydrase autoantibodies. Possible relevance of the presence of CA autoantibodies and clinical outcome is discussed.

Elimination of isocyanate and isothiocyanate molecules at the electrospray ionization ion trap, electrospray ionization quadrupole time-of-flight and matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry fragmentation of sodium cationized brassitin, brassinin and their glycosides.

Fragmentation mechanisms of phytoalexin analogs, including brassitin and brassinin and their glucosylated analogs, have been studied by electrospray (ESI) ion trap (IT) multistage (MS(n), n = 1-4) mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight (MALDI ToF/ToF) and ESI-Q/ToF tandem mass spectrometry techniques. At the fragmentation of sodium adducts a hitherto not described process has been elucidated The proposed mechanism of this process includes cyclization of the brassitin and brassinin cationized adducts through a six-membered cycle of the molecules and the elimination of isocyanate or isothiocyanate from the thio- or dithiocarbamate moiety, giving rise to [M + Na - 43](+) or [M + Na - 59](+) adducts. The elimination of NH=C=O or NH=C=S molecules has been confirmed by the high resolution measurement of the elemental composition of the ions produced and quantum-chemical calculations of the six-membered transition state. Other fragmentation routes include cleavage of an alkane linker, while numerous characteristic hexopyranose pathways are taking place in the glucosylated compounds. The presented theoretical data on the ESI and MALDI behavior of the saccharidic, as well as of the indole aglycon parts, can facilitate structural elucidation of the analogous compounds.

Detection of specific spectral markers of Coxiella burnetii isolates by MALDI-TOF mass spectrometry.

Specific markers for Coxiella burnetii (C.b.) isolates RSA 493, Priscilla, and BUD were detected using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). The method revealed noticeable differences in the ion signal profiles of the isolates in the mass range of 318 kDa. The number of characteristic ions for RSA 493, BUD, and Priscilla was 24, 15, and 7, respectively. The specific markers were compared against C.b. database using the Tag-Ident proteomics tool. For the isolates RSA 493, Priscilla and BUD there were identified 11, 5 and 3 potential biomarkers, respectively. This method represents a powerful tool for the rapid, sensitive, and differential characterization of C.b. isolates and is a good candidate for phyloproteomic approaches.

Efficiency of various serological techniques for diagnosing Coxiella burnetii infection.

An indirect immunofluorescence assay (IFA) using a recently developed commercial kit for detecting antibodies against Coxiella burnetii (C.b.), the etiological agent of Q fever, has been evaluated using human field serum samples. The IFA was compared with an ELISA and a complement fixation test (CFT). The IFA was based on the corpuscular C.b. phase I and phase II antigens specific to anti-C.b. phase I and II antibodies, respectively. Fifty sera from persons with symptoms of Q fever were examined in this study. The IFA compared with the ELISA showed the sensitivities of 97.7% and 87.2% for IgG and 66.7% and 60.0% for IgM phase II and I antibodies, respectively and the specificities of 100% and 90.0% for IgG and 75.9% and 64.7% for IgM phase II and phase I antibodies, respectively. Due to a limited number of sera positive in the IgA antibody testing, the data presented should be considered with caution. It appears that the IFA strikes a very good balance between high specificity and sensitivity with phase II and phase I IgG antibodies and a less satisfactory one with IgM antibodies. The CFT failed in one of the above aspects showing a good specificity but a poor sensitivity, especially for phase I antibodies. The study demonstrated that the IFA is suitable for diagnosing Q fever and its therapeutic follow-up and is a good candidate for screening sera in large numbers. A certain limitation, especially in testing early stages of the chronic disease, could be a low fluorescence intensity of the IgA positive control in comparison with the IgA negative one.

Initial peptide mass fingerprinting analysis of proteins obtained by lysis of Coxiella burnetii cells.

Whereas the complete genome of Coxiella burnetii (C.b.), the etiological agent of Q-fever, has recently been published (Seshadri et al., Proc. Natl. Acad. Sci. USA. 100, 5455-5460, 2003), the C.b. proteome is still under study. Using the bioinformatic approach, we found in total 309 proteins on two dimensional electroctrophoretic images of C.b. whole cell lysates. Eighteen major protein species were subjected to peptide mass fingerprinting and identified as the products of 6 known open reading frames (ORFs): the chaperone DnaK (heat shock 70 K protein), chaperonin 60 K (GroEL protein, heat shock protein B), DnaJ-like protein djlA (mucoidy activation protein mucZ), elongation factor Ts (EF-Ts), ribosomal protein L7/L12, and chaperonin 10 K (GroES protein, heat shock protein A).