A microtiter fluorometric assay to detect the germination of Bacillus anthracis spores and the germination inhibitory effects of antibodies.

Bacillus anthracis spore germination is usually detected in vitro by alterations in spore refractility, heat resistance, and stainability. We developed a more quantitative, sensitive, and semi-automated procedure for detecting germination by using a microtiter kinetic reader for fluorescence spectrophotometry. The procedure was based on the increase in fluorescence of spores with time during their incubation in germination medium containing a fluorescent nucleic acid-binding dye which stained germinated B. anthracis but not ungerminated (UG) spores. Spore germination in the presence of several germinants was characterized. Although L-alanine and inosine alone stimulated rapid germination in this assay, a medium containing optimal concentrations of L-alanine, adenosine, and casamino acids gave low background fluorescence, stimulated germination completely, and at a reasonable rate. Suspensions of heat-activated, UG spores of B. anthracis strain Ames were preincubated with antibodies (Abs) against whole spores to assess their effect on germination. Analyses of the germination data obtained revealed significant differences between spores pretreated with these Abs and those treated with non-immune sera or IgG. Germination inhibitory activity (GIA) was detected for several polyclonal rabbit anti-spore Ab preparations. These included anti-Ames strain spore antisera, IgG purified from the latter, and spore affinity-purified Abs from antisera elicited against four strains of B. anthracis. Abs elicited against UG as well as completely germinated Ames spores inhibited germination. Abs were ranked according to their GIA, and those specific for UG spores usually exhibited greater GIA. Direct binding to spores of these Abs was detected by an ELISA with whole un-germinated Ames spores. Although specific binding to spores by the anti-spore Abs was shown, their titers did not correlate with their GIA levels. Current efforts are focused on identifying the spore antigens recognized by the anti-spore Abs, characterizing the role of these targeted antigens in disease pathogenesis, and evaluating the ability of specific anti-spore Abs to protect against infection with B. anthracis.