Improved Quantification, Propagation, Purification and Storage of the Obligate Intracellular Human Pathogen <i>Orientia tsutsugamushi</i>

<div><p>Background</p><p>Scrub typhus is a leading cause of serious febrile illness in rural Southeast Asia. The causative agent, <i>Orientia tsutsugamushi</i>, is an obligate intracellular bacterium that is transmitted to humans by the bite of a <i>Leptotrombidium</i> mite. Research into the basic mechanisms of cell biology and pathogenicity of <i>O</i>. <i>tsutsugamushi</i> has lagged behind that of other important human pathogens. One reason for this is that <i>O</i>. <i>tsutsugamushi</i> is an obligate intracellular bacterium that can only be cultured in mammalian cells and that requires specific methodologies for propagation and analysis. Here, we have performed a body of work designed to improve methods for quantification, propagation, purification and long-term storage of this important but neglected human pathogen. These results will be useful to other researchers working on <i>O</i>. <i>tsutsugamushi</i> and also other obligate intracellular pathogens such as those in the <i>Rickettsiales</i> and <i>Chlamydiales</i> families.</p><p>Methodology</p><p>A clinical isolate of <i>O</i>. <i>tsutsugamushi</i> was grown in cultured mouse embryonic fibroblast (L929) cells. Bacterial growth was measured using an <i>O</i>. <i>tsutsugamushi</i>-specific qPCR assay. Conditions leading to improvements in viability and growth were monitored in terms of the effect on bacterial cell number after growth in cultured mammalian cells.</p><p>Key results</p><p></p><p></p><p>Development of a standardised growth assay to quantify bacterial replication and viability <i>in vitro</i>.</p><p></p><p></p><p>Quantitative comparison of different DNA extraction methods.</p><p></p><p></p><p>Quantification of the effect on growth of FBS concentration, daunorubicin supplementation, media composition, host cell confluence at infection and frequency of media replacement.</p><p></p><p></p><p>Optimisation of bacterial purification including a comparison of host cell lysis methods, purification temperature, bacterial yield calculations and bacterial pelleting at different centrifugation speeds.</p><p></p><p></p><p>Quantification of bacterial viability loss after long term storage and freezing under a range of conditions including different freezing buffers and different rates of freezing.</p><p></p><p></p><p>Conclusions</p><p>Here we present a standardised method for comparing the viability of <i>O</i>. <i>tsutsugamushi</i> after purification, treatment and propagation under various conditions. Taken together, we present a body of data to support improved techniques for propagation, purification and storage of this organism. This data will be useful both for improving clinical isolation rates as well as performing <i>in vitro</i> cell biology experiments.</p>