Detection of biological weapons is a primary concern in force protection, treaty verification, and safeguarding civilian populations against domestic terrorism. One great concern is the detection of Bacillus anthracis, the causative agent of anthrax. Assays for detection in the laboratory often employ inactivated preparations of spores or nonpathogenic simulants. This study uses several common biodetection platforms to detect B. anthracis spores that have been inactivated by two methods and compares those data to detection of spores that have not been inactivated. The data demonstrate that inactivation methods can affect the sensitivity of nucleic acid-and antibody-based assays for the detection of B. anthracis spores. These effects should be taken into consideration when comparing laboratory results to data collected and assayed during field deployment.Bacillus anthracis, the causative agent of anthrax, is an important zoonotic disease of domestic livestock. It is also associated with the waste from certain processing industries such as tanneries and can be found in the effluent from water processing plants (1). Cutaneous anthrax is associated with the handling of contaminated animal products and has a mortality rate of 5 to 20% when untreated. Inhalation of B. anthracis spores causes pneumonic anthrax that has a mortality rate approaching 100%. Antibiotic treatment is largely unsuccessful after the appearance of symptoms (11). From a military standpoint, B. anthracis has been described as the ultimate biological weapon because of its virulence and persistence on a battlefield when it is disseminated as a desiccated spore (13). Endospores are dormant forms of bacteria that are stable for great lengths of time and are resistant to inactivation from radiation and heat. Bacillus spores are so resistant and hardy that they have been revived from the abdominal cavity of an extinct bee entombed within Dominican amber 25 to 40 million years ago (2) and isolated from a brine inclusion dated at 250 million years old (14).Driven by the desire to develop and optimize detection devices to monitor and track the spores of B. anthracis, researchers face the need to handle moderate amounts of spore antigens. However, biosafety containment and occupational exposure are of great concern and necessitate inactivating the spores by gamma irradiation with a cobalt source or by thermal treatment such as autoclaving. B. anthracis has two major virulence factors encoded by plasmids pX01 (77 kb) and pX02 (95 kb). The plasmid pX01 codes for the lethal factor, the edema factor, and protective antigen that together form a tripartite protein exotoxin (4,7,9,12). Plasmid pX02 codes for the spore capsule (3). Laboratory detection assays often use inactivated spore preparations or nonpathogenic simulants to conduct development and testing of biosensors (6). The optimization of biodetection assays can necessitate handling moderate quantities of dangerous pathogens. Federal regulations limit the movement of pathogens and regulate safety controls...
The diagnosis of human cases of tularemia often relies upon the demonstration of an antibody response to Francisella tularensis or the direct culturing of the bacteria from the patient. Antibody response is not detectable until 2 weeks or more after infection, and culturing requires special media and suspicion of tularemia. In addition, handling live Francisella poses a risk to laboratory personnel due to the highly infectious nature of this pathogen. In an effort to develop a rapid diagnostic assay for tularemia, we investigated the use of TaqMan 5 hydrolysis fluorogenic PCR to detect the organism in tissues of infected mice. Mice were infected to produce respiratory tularemia. The fopA and tul4 genes of F. tularensis were amplified from infected spleen, lung, liver, and kidney tissues sampled over a 5-day period. The samples were analyzed using the laboratory-based Applied Biosystems International 7900 and the Smiths Detection-Edgewood BioSeeq, a hand-held portable fluorescence thermocycler designed for use in the field. A comparison of culturing and PCR for detection of bacteria in infected tissues shows that culturing was more sensitive than PCR. However, the results for culture take 72 h, whereas PCR results were available within 4 h. PCR was able to detect infection in all the tissues tested. Lung tissue showed the earliest response at 2 days when tested with the ABI 7900 and in 3 days when tested with the BioSeeq. The results were in agreement between the ABI 7900 and the BioSeeq when presented with the same sample. Template preparation may account for the loss of sensitivity compared to culturing techniques. The hand-held BioSeeq thermocycler shows promise as an expedient means of forward diagnosis of infection in the field.
Biological threat detection programs that collect air samples and monitor for large-scale release of biowarfare agents generate large numbers of samples that must be quickly and accurately screened for the presence of biological agents. An impediment to the rapid analysis of large numbers of environmental biological samples is that manual laboratory processes are time-consuming and require resources to maintain infrastructure, trained personnel, and adequate supplies of testing reagents. An ideal screening system would be capable of processing multiple samples rapidly, cost-effectively, and with minimal personnel. In the present study, we evaluated the Automated Biological Agent Testing System (ABATS) to explore the capability of automation to increase sample throughput, maximize system accuracy, and reduce the analysis costs associated with biological threat agent screening in environmental samples. This study demonstrates the utility of this concept and the potential of an automated system to address the growing environmental monitoring needs of the United States.
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