Isolation and confirmation of Bacillus anthracis from field outbreaks by conventional methods and PCR

The present study was carried out to isolate the anthrax organisms from different species, viz., sheep, goat, and cattle, from different geographical regions of Karnataka, viz., Gouribidanur taluk of Chikkaballapur district, Sira, Koratagere, Madhugiri, Chikkanayakanahalli, Pavagada taluk of Tumkur district, and Hagari Bommanahalli, Kottur taluk of Vijayanagar district, Kampli, Bellary, and Siraguppa taluks of Bellary district. Initially, the samples, such as ear pieces and blood samples collected from the dead animals, were streaked onto blood agar and later onto PLET agar for isolation and to study the microscopic and cultural characteristics. A total of twenty grayish-white colonies with a frosted glass-like appearance, without hemolysis, were observed on blood agar, and small rough, opaque colonies were observed on PLET agar. After the isolation of suspected Bacillus anthracis organisms, the pure cultures were used to confirm the isolates by conventional PCR by targeting the _rpo_B gene and Ba813 genes. The rpo_B is a chromosomal gene; amplicons of this gene will clearly indicate the positivity of the anthrax infection. Subsequently, the isolates were further tested for the presence of the Ba813 gene to confirm the disease. 175 bp and 152 bp amplicons were observed on agarose gel for the rpo_B and Ba813 genes, respectively.