Characterization of Bacillus anthracis replication and persistence on environmental substrates associated with wildlife anthrax outbreaks

Anthrax is a zoonosis caused by the environmentally maintained, spore-forming bacterium Bacillus anthracis, affecting humans, livestock, and wildlife nearly worldwide. Bacterial spores are ingested, inhaled, and may be mechanically transmitted by biting insects or injection as occurs during heroin-associated human cases. Herbivorous hoofstock are very susceptible to anthrax. When these hosts die of anthrax, a localized infectious zone (LIZ) forms in the area surrounding the carcass as it is scavenged and decomposes, where viable populations of vegetative B. anthracis and spores contaminate the environment. In many settings, necrophagous flies contaminate the outer carcass, surrounding soils, and vegetation with viable pathogen while scavenging. Field observations in Texas have confirmed this process and identified primary browse species (e.g., persimmon) are contaminated. However, there are limited data available on B. anthracis survival on environmental substrates immediately following host death at a LIZ. Toward this, we simulated fly contamination by inoculating live-attenuated, fully virulent laboratory-adapted, and fully virulent wild B. anthracis strains on untreated leaves and rocks for 2, 5, and 7 days. At each time point after inoculation, the number of vegetative cells and spores were determined. Sporulation rates were extracted from these different time points to enable comparison of sporulation speeds between B. anthracis strains with different natural histories. We found all B. anthracis strains used in this study could multiply for 2 or more days post inoculation and persist on leaves and rocks for at least seven days with variation by strain. We found differences in sporulation rates between laboratory-adapted strains and wild isolates, with the live-attenuated strain sporulating fastest, followed by the wild isolates, then laboratory-adapted virulent strains. Extrapolating our wild strain lab results to potential contamination, a single blow fly may contaminate leaves with up to 8.62 x 10(5) spores per day and a single carcass may host thousands of flies. Replication outside of the carcass and rapid sporulation confirms the LIZ extends beyond the carcass for several days after formation and supports the necrophagous fly transmission pathway for amplifying cases during an outbreak. We note caution must be taken when extrapolating replication and sporulation rates from live-attenuated and laboratory-adapted strains of B. anthracis.