RESEARCH ARTICLE Cell-Free Propagation of Coxiella burnetii Does Not Affect Its Relative Virulence

Q fever is caused by the obligate intracellular bacterium Coxiella burnetii. In vitro growth of the bacterium is usually limited to viable eukaryotic host cells imposing experimental con-straints for molecular studies, such as the identification and characterisation of major viru-lence factors. Studies of pathogenicity may benefit from the recent development of an extracellular growth medium for C. burnetii. However, it is crucial to investigate the consis-tency of the virulence phenotype of strains propagated by the two fundamentally different culturing systems. In the present study, we assessed the viability of C. burnetii and the lipo-polysaccaride (LPS) encoding region of the bacteria in both culture systems as indirect but key parameters to the infection potential of C. burnetii. Propidium monoazide (PMA) treat-ment-based real-time PCR was used for enumeration of viable C. burnetii which were vali-dated by fluorescent infectious focus forming unit counting assays. Furthermore, RNA isolated from C. burnetiipropagated in both the culture systems was examined for LPS-related gene expression. All thus far known LPS-related genes were found to be expressed in early passages in both culturing systems indicating the presence of predominantly the phase I form of C. burnetii. Finally, we used immune-competent mice to provide direct evi-dence, that the relative virulence of different C. burnetii strains is essentially the same for both axenic and cell-based methods of propagation