Molecular analysis of Bacillus anthracis isolates from Karnataka's ruminant anthrax outbreaks reveals genetic relationships and environmental factors influencing spore persistence.

Background: Anthrax outbreaks continue to be reported from several districts of Karnataka in India. Many outbreaks in livestock and humans have reported, the soil and climatic conditions would have supported long-term spore persistence. Hence, the state of Karnataka in India was chosen as an initial pilot site to study, isolate, and characterize Bacillus anthracis. In this study, we integrate molecular typing and soil analysis across endemic and non-endemic areas, an approach that has been scarcely explored and could be a novel contribution. By comparing both endemic and non-endemic regions, it would be an opportunity to examine soil-related ecological factors that could have been linked to the anthrax outbreaks. A total of 45 suspected animal anthrax cases were investigated using culture and PCR-based methods. Colonies with Medusa head-like appearance on blood agar plates were subjected to PCR assays targeting protective antigen (pXO1), capsular gene (pXO2), and the chromosomal rpoB genes. Additionally, soil samples from 12 different sites (six endemic and six non-endemic) were processed using the Ground Anthrax Bacillus Refined Isolation (GABRI) method, which was used to enhance the detection of B. anthracis spores by reducing environmental contaminants that may inhibit spore germination and growth.

Results: Out of 45 suspected animal anthrax cases, 09 isolates were confirmed as Bacillus anthracis with colony characteristics of greyish-white, frosted-glass on blood agar, further verified through PCR analysis. Phylogenetic analysis based on the rpoB gene demonstrated a close genetic relationship among these isolates, suggesting that the transmission of spores was localized and likely facilitated by animal movement. The soil analysis showed that the endemic sites had an alkaline pH of 7.81-8.9, higher organic carbon 0.45-4.36 %, elevated phosphorus 10.32 to 123.7 kg/ha, and greater clay content up to 45 % in Bellary, contributing to higher survivability and retention of spores in endemic regions. In contrast, non-endemic soil exhibited neutral to slightly acidic pH (6.1-6.85), lower phosphorus levels, and sandy clay loam texture that may limit long-term spore retention.

Conclusion: The study offers significant insights into the environmental and genetic factors contributing to the persistence of Bacillus anthracis in both endemic and non-endemic regions. The finding shows that soil plays a crucial role in the survival and transmission of anthrax spores, with higher retention observed in endemic areas. The close genetic relationship among isolates further suggests localized transmission, likely influenced by animal movement. These results underline the need for continued surveillance and prevention strategies, especially in regions with a history of anthrax outbreaks.