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

Authors: Rothrock, Michael; Cook, Kimberly - Kim; Lovanh, Nanh; Warren, Jason; Sistani, Karamat

Submitted to: Poultry Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/26/2008
Publication Date: 7/8/2008
Citation: Rothrock Jr, M.J., Cook, K.L., Lovanh, N.C., Warren, J.G., Sistani, K.R. 2008. Development of a Quantitative Real-Time Polymerase Chain Reaction Assay to Target a Novel Group of Ammonia-Producing Bacteria Found in Poultry Litter . Poultry Science. 87(6):1058-1067

Interpretive Summary: Ammonia emissions from poultry litter have implications for both flock health and performance, as well as for air and water quality. Numerous amendments can be added to the litter once ammonia has already been produced in order to reduce the emissions from the poultry houses. Very little work has dealt with targeting the microbes responsible for the ammonia production, and an understanding of these organisms can lead to treatment methods that stop ammonia before it is produced by the microbes, rather than afterwards. In this study, we targeted the microbes in chicken litter using a gene for the enzyme that is responsible for this ammonia production, urease. We developed a quantitative real-time PCR assay to specifically target a novel, dominant group of ammonia producing microbes found only in poultry litter. Using this assay, we found that these organisms are a significant portion of the overall bacterial population, and that the concentrations of these organisms were affected by both the chemical and physical parameters of the litter. Future applications of this assay include determining the efficacy of common litter amendment practices to determine how they affect the ammonia producing microbial population in the litter, as well as assessing novel and cost effective treatments to reduce ammonia emissions from poultry litter.

Technical Abstract: 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. The urease enzyme is responsible for the final step in the conversion of organic N (specifically uric acid and urea) to ammonia. Analysis of ureC (alpha subunit of the urease enzyme) from poultry litter revealed the presence of a novel, dominant group of ureolytic microbes. To target this group, specific primers and probe were designed and a new quantitative real-time PCR assay was developed. The assay allowed for the detection of ' 1 x 104 ureC target sequences (representing ' 10 copies per PCR reaction) and amplified target poultry litter and standard ureC samples with equally high efficiency over 8 orders of magnitude. In comparing a variety of pure cultures and environmental samples shown to possess urease activity, target ureC amplification was found only from poultry litter samples, demonstrating the specific nature of this assay. The concentration of cells containing the targeted ureC sequence ranged from 9x106 to 4x108 ureC cells per g litter, and accounted for 0.1-3.0% of the total bacterial population in the poultry litter. Different litter types from three different states were analyzed physically, chemically and biologically to determine which environmental parameters most affected the concentration of this targeted ureC group. Based on Principal Component Analysis, approximately 40% of the variance in the concentrations of ureC cells was due to the chemical makeup of the litter (organic-N, total N, total C), where as an additional 30% of the variance was accounted for by the physical makeup of the litter (litter composition, moisture content, pH). Future applications of this assay could include determining the efficacy of current ammonia reducing litter amendments or in designing more efficient treatment protocols.