Isolation and identification of avirulent strains of Bacillus anthracis from environmental samples in Central Java, Indonesia

Background and Aim: Anthrax is a non-contagious infectious disease caused by Bacillus anthracis. The bacteria form
spores that are resistant to extreme conditions and can contaminate the environment for decades. This study aimed to detect and characterize B. anthracis found in endemic areas of anthrax in Yogyakarta and Central Java province, Indonesia.

Materials and Methods: Soil samples were collected from Gunungkidul regency, Yogyakarta province (n=315) and
Boyolali regency,  Central Java province (n=100). Additional soil samples (n=10) and straw samples (n=5) were obtained
from Pati regency, Central Java province. The isolation and identification of B. anthracis were performed using conventional methods: Morphology of bacteria colony in solid media, Gram staining, capsule staining, spores staining, and motility test. Isolates were further identified using polymerase chain reaction (PCR) against Ba813, lef (pXO1), and capC (pXO2) gene. An avirulent vaccine strain of B. anthracis (strain 34F2) was used as a control.

Results: Only four samples grew on blood agar with a ground-glass appearance, white-gray colony (Gunungkidul and
avirulent strain) or yellowish (Boyolali and Pati). All were Gram-positive, presented chains, square-ended rods, spores,
and were then identified as B. anthracis. Boyolali, Pati, and avirulent strain isolates had slightly different characteristics, including the growth of non-mucoid in the bicarbonate agar medium, and their uncapsulated form. The PCR showed two Gunungkidul isolates which amplified three genes, including Ba813, lef, and capC. Contrarily, the other isolates did not amplify the capC gene.

Conclusion: Gunungkidul isolates were identified as virulent strains of B. anthracis while Boyolali and Pati isolates were proposed as avirulent strains. This is the first report of isolation and identification of avirulent strains of B. anthracis in Central Java, Indonesia