Somatic soluble antigen was prepared by culturing Lm in tryptic soy broth for 18 h, washing it in PBS, and subsequently subjecting it to ultrasonication.1 g (wet weight) of bacterial cells were suspended in 10 ml of PBS and sonicated five times for 1 min (87.5%, output, degree 7 on a sonifier model S-125; Branson Sonic Power, USA) on ice. The sonicated suspension was centrifuged at 39 000 U for 50 min, and the supernatant was filter sterilized (pore size,0.45 mm) and stored at 220uC at a dilution of 1:100 in PBS [42] .