Fluorometric Assay to Detect the Germination of Bacillus Anthracis Spores and the Germination Inhibitory Effects of Antibodies

A sensitive assay for germination of spores of the anthrax agent was developed using a fluorescence reader. Significant inhibition of germination was detected for spores initially treated with antispore or antitoxin antibodies. Antigens specific to ungerminated spores that stimulate potentially protective antibodies are being identified and their role in inhibition of germination and in immune protection against anthrax will be determined. The germination of spores of Bacillus anthracis is typically detected by alterations in spore refractility, stainability, and absorbance (OD560). We have developed a more quantitative, semi-automated procedure for detecting germination by using a microtiter kinetic reader for fluorescence spectrophotometry (Biotek FL600). The procedure was based on the increase in fluorescence of spores with time during their incubation in germination medium containing Syto-9, a fluorescent nucleic acidbinding dye which stained germinated B. anthracis but not dormant spores. A colorless, defined medium containing equal parts of L-alanine, adenosine, and casamino acids provided low levels of background fluorescence, stimulated germination, and yielded reasonable germination kinetics. Antibodies (Abs) to the protective antigen (PA) component of the anthrax toxins of B. anthracis have been shown to inhibit in vitro germination. Regression analyses of the germination data obtained with the reader yielded parameters (i.e., the difference between the baseline and maximal fluorescence values [a coefficient] and the area under the curve) that gave significant differences between spores pretreated with anti-PA or antispore antibodies and buffer or nonimmune sera. We are testing sera from vaccinated or passively-immunized animals to determine the correlation between serum germination inhibitory activity, titers of serum anti-PA or antispore antibodies, and survival after lethal challenge. INTRODUCTION The recent occurrence of 22 cases of bioterrorism-associated anthrax has confirmed the known potential of Bacillus anthracis to be used as a biological weapon. Early detection and aggressive intervention with antibiotics resulted in the survival of 6 of the 11 patients with the usually lethal inhalational form of anthrax. However, infected and/or exposed individuals treated with antibiotics are at risk of developing inhalational anthrax when the therapy is terminated and dormant spores are able to germinate. The protection of individuals exposed to the spores might be enhanced by preventing germination of the infectious spores into the replicating bacilli. The bacilli produce the anthrax edema and lethal toxins, composed of the cell-