Recently we evaluated the performance of a new liquid culture method, the TREK ESP para-JEM Culture System II, as compared with our standard method. The ESP system performed well with a strong reduction in time to detection (TTD). The LJ system, however, demonstrated a higher sensitivity for detection of low shedders. We also observed that weekly shaking of liquid cultures enhanced growth. To optimize the diagnostic performance of the ESP system, we evaluated three decontamination methods, combined with either stationary incubation or weekly shaking, on fresh bovine fecal samples from - predominantly light - shedders and from culture negative cattle. These samples were also cultured on LJ agar slants. We confirmed in this study the previously observed reduction in TTD by weekly shaking using the prescribed decontamination method. No reduction, however, was observed for the in-house and Stabel decontamination method. Mean TTD was lowest for the in-house decontamination method. With the latter method, however, we found surprisingly high numbers of false-positives. In another experiment, fresh fecal samples (n=116) originating from four infected herds were cultured in parallel on LJ agar slants and in ESP para-JEM culture bottles after decontamination as described by Stabel et al. From one infected herd also pooled samples were investigated. Both culture systems scored 40 samples positive with 32 samples in common. The mean TTD for the ESP system (39.1 days) was significantly lower than for the LJ system (75.6 days). For pooled samples from one infected herd a good agreement was found between both culture systems and individual LJ culture results. In conclusion, the ESP System yielded equivalent or slightly better results than the standard LJ system. The Stabel decontamination method combined the advantages of low numbers of false-positives with a relatively low TTD after stationary incubation.