Bacillus Spore Inactivation Methods Affect Detection Assays

Abstract

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...

Research and Development of On-site Decontamination System for Biological and Chemical Warfare Agents

“…However, existence of the decontaminating reagents in the samples may interfere with the accurate quantification. 66,86) …”

<i>Bacillus pumilus</i>: Possible Model for the Bioweapon <i>Bacillus anthracis</i>

“…With this medium, B. thuringiensis yielded the lowest number of spores and required 16 days to achieve 90% sporulation. Successful sporulation of B. thuringiensis has been reported using nutrient broth yeast extract agar [9], T-soy agar supplemented with 5% sheep blood [6] and sporulation medium S [18].…”

“…There are risks associated with working with B. anthracis and inactivation of spores by heat, chemicals or radiation may alter antigenicity [6]. Many detection assays are immunologically centered.…”

“…Due to the virulent nature of B. anthracis, strict adherence to safety protocols must be followed. Inactivation of spores from B. anthracis may result in alterations in antigenic structures or changes in targeted nucleic acid sequences that may affect detection [6]. To permit more thorough investigation of possible detection methodologies, it is often desirable to use spores from other Bacillus spp.…”

ELISA Detection of Francisella tularensis using Polyclonaland Monoclonal Antibodies

“…The enzyme-linked immunosorbent assay (ELISA) has been proved as a reliable technique for the diagnosis of several serious diseases and detection of causative agents. Sera from patient suffering with anthrax were evaluated using ELISA 8 method; the whole Bacillus anthracis cells were detected using ELISA and compared with PCR and flow cytometry 9 . Agglutination test and ELISA were used for serological testing of sera from patients suffered from brucellosis 10 and antibodies against Brucella melitensis were detected in sheep milk 11 .…”