Enumeration of Megasphaera elsdenii in rumen contents by real-time Taq nuclease assay

Aims: To develop a real-time Taq nuclease assay (TNA) to enable the in vivo enumeration of Megasphaera elsdenii. Methods and Results: Megasphaera elsdenii YE34 was phenotypically characteristic of the species and had 16S rDNA sequence similarity of 98% to previously described isolates. Calibration of the number of cells of M. elsdenii against the cycle threshold of fluorescent dye release gave a straight-line relationship with a correlation coefficient approximating unity. The specificity of the assay for M. elsdenii was confirmed by performing it against a panel of 24 heterogenous, mainly ruminal bacteria. Megasphaera elsdenii was not detected in ruminal contents from a pasture-fed steer but was readily detected 2 and 50 h after the probiotic introduction of the bacterium into the rumen. Conclusions: Real-time TNA has provided a sensitive and specific means of enumerating the M. elsdenii population in rumen contents. Significance and Impact of the Study: Megasphaera elsdenii is an important lactate-degrading ruminal bacterium that has been selected for probiotic use to prevent acidosis and enhance starch utilization in grain-fed cattle. The assay developed in this study provides a tool for determining the ability of probiotically-introduced M. elsdenii to establish useful populations in the rumen.