Rapid bioassay for detection of Bacillus anthracis in mice

A rapid-bioassay method (RBA) of diagnosing anthrax was developed in mice. A standard RBA consisted of intraperitoneal inoculation of a suspected culture or of a suspension of raw infected animal tissues in four pairs of mice. At 60, 120 and 180 min after inoculation, two mice were killed and smears and impression smears were made from the peritoneal fluid, heart blood, spleen, liver, and kidneys, and then fixed, stained and microscopically examined. Encapsulated rods seen in microscopy confirmed the diagnosis. The remaining pair of mice was kept for a 10-day observation period and served as a control. In the experiments described in this study, RBA showed a high efficiency. A vegetative culture of 18-20 h of Bacillus anthracis growth demonstrated encapsulation in mice as early as 120 min after inoculation; a culture of 3-5 or 6-12 h of growth showed encapsulation at 60 min. Old spores gave encapsulation at 180 min, but, if previously plated for 6 h, encapsulation was observed as early as 60 min after inoculation. Inoculation of raw infected tissues gave positive results at 120 min. As compared with the challenged controls, no deviations between the results of RBA and the final diagnosis of anthrax were observed, but RBA appears to be 7-17 times more rapid. Some variations of the standard RBA are also suggested.