Infection of Human Monocyte-Derived Macrophages With Coxiella burnetii - Bacterial Pathogenesis: Methods and Protocols - page 179

C. burnetii does not appear to cause overt disease with the exception of sheep
and goats, where it can cause abortions (4) . Unlike other obligate intracellular
bacteria, C. burnetii is remarkably resistant to desiccation and therefore can
persist in contaminated soils for extended periods (2) . Inhalation of contami-
nated aerosols is the primary route of human infection with C. burnetii (5) , and
disease can be initiated in both humans and animals by less than 10 organisms
(6,7) . Consequently, C. burnetii can represent an occupational hazard, partic-
ularly for individuals involved in animal husbandry operations where large
numbers of organisms can be shed into the environment during parturition (8) .
As an obligate intracellular bacterium, C. burnetii requires a viable eucaryotic
host cell for propagation. Laboratory cultivation of C. burnetii has been accom-
plished in animals (primarily guinea pigs and mice), embryonated hen's eggs,
and tissue culture cells (5) . Regardless of the host and cell type, C. burnetii
replicates in a membrane-bound vacuole with lysosomal characteristics (9) .
Cell culture is currently the method of choice for propagation of C. burnetii .
A variety of primary and continuous cell lines support vigorous growth of
the organism including primary chick and mouse embryo fibroblasts and
continuous cell lines, like Vero (African green monkey kidney epithelial), BHK-
21 (hamster kidney fibroblast), L-929 (murine fibroblast), J774.1 and P388D1
(murine macrophage-like), and THP-1 (human monocyte-like) (1,10,11,12) .
Organisms are typically harvested when large vacuoles filled with C. burnetii
are observed throughout the cell culture ( 6-8 days post-infection). Cells are

scraped from culture flasks and mechanically disrupted to release intracellular
bacteria (13) . Purification of C. burnetii from host cell lysates involves a series
of differential centrifugation steps followed by density gradient centrifugation
through sucrose or RenoCal-76 (13,14,15) . The hydrophobic, truncated LPS
of phase II C. burnetii results in adherence of host material that consequently
results in lower yields of this strain when compared to yields of phase I
organisms producing full-length LPS (15) .
In vivo, C. burnetii primarily targets cells of the mononuclear phagocyte
system such as alveolar macrophages of the lung and Kupffer cells of the liver
(16) . Thus, primary human macrophages represent the most physiologically
relevant in vitro system to study Coxiella -host interactions. Here, we describe
protocols for quantifying infection of human peripheral blood monocyte-derived
macrophages (MDMs) by purified C. burnetii .

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2. Materials
2.1. Coxiella burnetii Propagation and Purification
1. RPMI medium with Glutamax I (Invitrogen, Carlsbad, CA) supplemented with
10 or 2% fetal bovine serum (FBS) (Invitrogen). Store at 4 °C.