For the toxin end point titer assay, Vero cells were seeded into a 96 wells plate at a density of 1610 4 cells per well and incubated overnight at 37uC and 5% CO 2 . The filter sterilized supernatants of 5, 8, 12, 24 and 48 hours post inoculation (hpi) were diluted 2, 10 1 , 10 2 , 10 3 , 10 4 and 10 5 fold in cell culture medium (Dulbecco modified Eagle medium (Lonza) supplemented with penicillin 100 u/mL, streptomycin 100 U/mL, fetal calf serum(10%). Fifty ml of the dilutions were added onto the Vero cell monolayers and incubated for 1 hr at 37uC and 5% CO 2 . For the neutralization assay a 2-fold dilution of each tested time point (5, 8, 12, 24 and 48 hpi) was pre-incubated with a 1/100 diluted anti-toxin (Techlab) for 1 hr at 37uC and 5% CO 2 . After the pre-incubation, 50 ml was added onto the Vero cell monolayers. The incubated bacterial supernatants were aspirated off after one hour and replaced with 100 ml cell culture medium. After 24 hrs of incubation the end-point titer was determined of each diluted time point [26] . The end-point titer was defined as the first dilution at which the Vero Cell morphology was indistinguishable from the neutralized 2-fold diluted supernatants [26] . The enzyme immunoassay (Ridascreen, Biopharma) was performed according manufacture's protocol.