anthracis and contaminants isolated by GABRI method Total of 10 Inhibitor Library MK 8931 order plates Total of 10 plates Total of 10 plates Undiluted

1:10 1:100 Undiluted 1:10 1:100 CFU of B. anthracis CFU of contaminants Faridpur 0 4 8 8482 2190 314 394 1622 Sapatul 108 32 0 1380 162 22 256 200 Dhunot 0 0 0 4404 598 60 10 1164 Santhia 120 128 15 4968 826 90 10,000 276 Shahazadpur 0 0 0 1074 100 14 10 280 Ullapara 20 0 0 66 2 0 68 130 Shahazadpur 2 0 0 426 44 2 12 176 Average 35.7 23.4 3.3 2971.4 560.3 71.7 1535.7 549.7 Classic method for isolation of B. anthracis The method used for the isolation of spores from environmental samples was that described in OIE Terrestrial Manual 2012 [15], with some modifications. For culturing and isolation of B. anthracis the TSMP medium was used, consisting in the semi-selective Columbia blood agar added MEK activity with trimethoprim (16 mg/lt), sulfamethoxazole (80 mg/lt), methanol (5 ml/lt) and polymyxin (300,000 units/lt). Based on our experience, TSMP has the same efficacy of PLET in isolating B. anthracis (data not shown). Briefly, to each 7.5 gram aliquot of soil sample were added 22.5 ml of deionized sterile water. After 30 minutes of washing by vortexing, the suspension was incubated at 64°C for 20 min to eliminate any vegetative forms of soil contaminants [16]. From each sample, 10 ml of supernatant were collected and dilutions of 1:10 and 1:100 were made

using normal saline solution. Subsequently, 10 plates of TMSP were seeded with the undiluted suspension (100 μl/plate), 10 plates with the 1:10 dilution and 10 plates with the 1:100 dilution. After 24 and 48 hours of incubation at 37°C, each plate was examined for the presence of suspect colonies of B. anthracis

and of contaminants. All colonies were counted. B. anthracis colonies were identified by Gram staining, colony morphology and anthrax-specific PCRs [17]. Ground anthrax bacillus refined isolation (GABRI) procedure To each 7.5 gram aliquot were added 22.5 ml of washing buffer consisting of deionized water containing 0.5% Tween 20. After 30 minutes of washing by vortexing, the suspension was centrifuged at 2000 rpm for 5 min to eliminate gross debris. The Low-density-lipoprotein receptor kinase supernatant was harvested and then incubated, aerobically, at 64°C for 20 min to eliminate vegetative forms of B. anthracis. After incubation, 5 ml of supernatant were added to 5 ml of Tryptose Phosphate Broth containing 125 μg/ml of Fosfomycin. Then, from each sample, 10 plates of TMSP were seeded with 1 ml/plate of the mix and were incubated, aerobically, at 37°C. After 24 and 48 hours of incubation, each plate was examined and the colonies of B. anthracis and of contaminants were counted. B. anthracis colonies were identified by anthrax-specific PCRs [17]. Statistical analysis The comparison between GABRI and standard methods, applied to the soil samples artificially and naturally contaminated, was carried out using the method of Bland and Altman [18].