Development of a Quantitative Real-Time Polymerase Chain Reaction Assay to Target a Novel Group of Ammonia-Producing Bacteria Found in Poultry Litter

Ammonia production in poultry houses has serious implications for flock health and performance, nutrient value of poultry litter, and energy costs for running poultry operations. In poultry litter, the conversion of organic N (uric acid and urea) to NH₄-N is a microbially mediated process. The urease enzyme is responsible for the final step in the conversion of urea to NH₄-N. Cloning and analysis of 168 urease sequences from extracted genomic DNA from poultry litter samples revealed the presence of a novel, dominant group of ureolytic microbes (representing 90% of the urease clone library). Specific primers and a probe were designed to target this novel poultry litter urease producer (PLUP) group, and a new quantitative real-time PCR assay was developed. The assay allowed for the detection of 10² copies of target urease sequences per PCR reaction (approximately 1 x 10⁴ cells per gram of poultry litter), and the reaction was linear over 8 orders of magnitude. Our PLUP group was present only in poultry litter and was not present in environmental samples from diverse agriculutural settings. This novel PLUP group represented between 0.1 to 3.1% of the total microbial populations (6.0 x 10⁶ to 2.4 x 10⁸ PLUP cells per gram of litter) from diverse poultry litter types. The PLUP cell concentrations were directly correlated to the total cell concentrations in the poultry litter and were found to be influenced by the physical parameters of the litters (bedding material, moisture content, pH), as well as the NH₄-N content of the litters, based on principal component analysis. Chemical parameters (organic N, total N, total C) were not found to be influential in the concentrations of our PLUP group in the diverse poultry litters Future applications of this assay could include determining the efficacy of current NH₄-N-reducing litter amendments or in designing more efficient treatment protocols.