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.

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.

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.