Journal article
Isolation from Animal Tissue and Genetic Transformation of Coxiella burnetii Are Facilitated by an Improved Axenic Growth Medium
Applied and environmental microbiology, Vol.77(11), pp.3720-3725
06/2011
Handle:
https://hdl.handle.net/2376/112670
PMCID: PMC3127619
PMID: 21478315
Abstract
We recently described acidified citrate cysteine medium (ACCM), which supports host cell-free (axenic) growth of
Coxiella burnetii
. After 6 days of incubation, greater than 3 logs of growth was achieved with the avirulent Nine Mile phase II (NMII) strain. Here, we describe modified ACCM and culture conditions that support improved growth of
C. burnetii
and their use in genetic transformation and pathogen isolation from tissue samples. ACCM was modified by replacing fetal bovine serum with methyl-β-cyclodextrin to generate ACCM-2. Cultivation of NMII in ACCM-2 with moderate shaking and in 2.5% oxygen yielded 4 to 5 logs of growth over 7 days. Similar growth was achieved with the virulent Nine Mile phase I and G isolates of
C. burnetii
. Colonies that developed after 6 days of growth in ACCM-2 agarose were approximately 0.5 mm in diameter, roughly 5-fold larger than those formed in ACCM agarose. By electron microscopy, colonies consisted primarily of the
C. burnetii
small cell variant morphological form. NMII was successfully cultured in ACCM-2 when medium was inoculated with as little as 10 genome equivalents contained in tissue homogenates from infected SCID mice. A completely axenic
C. burnetii
genetic transformation system was developed using ACCM-2 that allowed isolation of transformants in about 2 1/2 weeks. Transformation experiments demonstrated clonal populations in colonies and a transformation frequency of approximately 5 × 10
−5
. Cultivation in ACCM-2 will accelerate development of
C. burnetii
genetic tools and provide a sensitive means of primary isolation of the pathogen from Q fever patients.
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Details
- Title
- Isolation from Animal Tissue and Genetic Transformation of Coxiella burnetii Are Facilitated by an Improved Axenic Growth Medium
- Creators
- Anders Omsland - Coxiella Pathogenesis Section, Laboratory of Intracellular ParasitesPaul A Beare - Coxiella Pathogenesis Section, Laboratory of Intracellular ParasitesJoshua Hill - Coxiella Pathogenesis Section, Laboratory of Intracellular ParasitesDiane C Cockrell - Coxiella Pathogenesis Section, Laboratory of Intracellular ParasitesDale Howe - Coxiella Pathogenesis Section, Laboratory of Intracellular ParasitesBryan Hansen - Electron Microscopy Unit, Research Technologies Branch, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana 59840James E Samuel - Department of Microbial and Molecular Pathogenesis, Texas A&M Health Sciences Center, College Station, Texas 77843Robert A Heinzen - Coxiella Pathogenesis Section, Laboratory of Intracellular Parasites
- Publication Details
- Applied and environmental microbiology, Vol.77(11), pp.3720-3725
- Academic Unit
- Paul G. Allen School for Global Animal Health
- Publisher
- American Society for Microbiology; 1752 N St., N.W., Washington, DC
- Identifiers
- 99900547970301842
- Language
- English
- Resource Type
- Journal article