Development of a Quantitative TaqMan{trademark}-PCR Assay and Feasibility of Atmosphoric Collection for Coccidioides Immits for Ecological Studies

Understanding and then modeling the complex relationships between environmental, climatological, and biological systems can present great opportunities for developing effective strategies to prevent outbreaks of environmentally linked infectious disease. For example, a report from the American Academy of Microbiology (Rose et al., 2001) points out that including climatic signals and ecological triggers into the decision making process for disease management, is going to be essential for developing a proactive approach for protecting public health, and circumventing the practice of having to wait for clinical cases to appear before any action is taken. To meet this grand challenge, specific and sensitive analytical techniques and effective collection devices are required for finding the microorganisms of concern in environmentally relevant media (air, soil, water, vegetation, etc.), and a multidisciplinary approach is needed for evaluating all of the environmental and biological data and building the applicable predictive models. In this study, the unique capabilities in biotechnology and environmental science available at Lawrence Livermore National Laboratory (LLNL) are combined with the distinguished and highly regarded expertise for clinically investigating and treating infectious disease at the Department of Microbiology and Immunology in the School of Medicine of the University of California, Davis, in order to develop, more » standardize, validate, and test safely the feasibility of applying advanced polymerase chain reaction (PCR) technology and new air-sample collection media that would be appropriate for addressing comprehensively the environmentally linked, medically important infectious disease Valley Fever (coccidioidomycosis). The responsible agent for this disease is the airborne spore (arthroconidia) of the pathogenic fungus Coccidioides immitis, which is a microorganism that is endemic to California, Arizona, and the southwestern United States, and also is identified as a select (biological) agent in the federal Anti-Terrorist and Effective Death Penalty Act. Successful demonstration of these tools in this study will place this multidisciplinary team in a credible position to proceed with additional research designed to determine the climatic signals and ecological triggers that would be associated with the presence of this microorganism environmentally and that would correlate with subsequent outbreaks of Valley Fever clinically. Results from such future research would then provide the information needed for environmental intervention of the disease occurrence, well before clinical cases appear. The technology and modeling developed for such a study also could be used for determining the ecology of other environmentally linked, medically important infectious diseases that occur naturally or that might be introduced deliberately into environmental media indoors or outside. The following approach was taken to achieve the technological objectives of this study. First, the protocols for the TaqMan{trademark}-PCR assay were enhanced to achieve the superior specificity and sensitivity required for quantifying, from a DNA signature, those C. immitis spores that are present in calibration samples (consisting of known quantities of pure culture inoculated onto air-filter concentrate, and then removed, and the DNA extracted) and those that are present within the calibration range on air filters obtained from the field and handled similarly. Second, the feasibility of using advanced nuclepore air-filter media to collect the spores from ambient air in C immitis endemic areas in the Central Valley of California was evaluated. These membranes permit the physics of high-volume air sampling to be used to filter larger amounts of air than previously possible for detecting airborne microorganisms. Thus, the higher volume of air flow improves the likelihood of capturing on the filter any C. immitis spores resuspended from nearby soil, where this microorganism grows prior to sporulation, and where its exact location can be elusive under any environmental conditions. Finally, experiments were conducted in the Department of Microbiology and Immunology at the School of Medicine of the University of California, Davis (UCD), to demonstrate that C. immitis spores can be killed, whether in pure culture or on air-filter media, to ensure safe handling during laboratory processing and analyses. « less