PCR detection of the pyridinediol-degrading ruminal bacterium, Synergistesjonesii, in the rumen fluid of cattle

Synergistes jonesii, a Gram-negative rumen bacterium, can catabolize a toxi c pyridinediol, 3-hydroxy-4-pyridone, which is produced from mimosine prese nt in Leucaena fodder in the rumen. Thus, it protects ruminants from the ha rmful effects of this toxin. A PCR-based method was developed to detect the presence of this pyridinediol-degrading bacterium in the rumen fluid of ca ttle. A unique 0.9-kb DNA sequence was identified by sequencing several fra gments from a genomic library of S, jonesii strain 78-1 and four pairs of p rimers were designed and used for PCR amplification of specific fragments. The PCR conditions were optimized with one pair of primers that amplified a 349-bp fragment from S. jonesii DNA but not from any other bacterial DNA t ested. This method could detect the presence of 1 to 10 bacteria in a sampl e. The method was tried on rumen fluid samples added with a known number of the bacterium With a simple pretreatment of the mixed sample, the PCR meth od reached a sensitivity only 100 fold lower than with pure-culture samples . Application of this method detected the presence of S. jonesii in some of the rumen fluid samples from cattle in Hawaii. This method will be a valua ble tool to determine the effects of inoculation of cattle with S, jonesii and to monitor the population dynamics of this pyridinediol-degrading bacte rium in cattle fed with different amounts of Leucaena.