2011 Annual Report
1a.Objectives (from AD-416)
The objectives of this research project are to conduct a survey of bacteria in dairy wastewaters utilizing a polymerase chain reaction (PCR)-based approach, then quantify select pathogens using quantitative PCR and traditional culture-based (plate counting) methods. Indicator organisms will also be quantified in the wastewaters to determine if a relationship exists between the indicator organism and pathogen concentrations.
1b.Approach (from AD-416)
During year 1, samples will be collected from as many as 10 dairy lagoons. At each lagoon, a total of 8 samples (250 mL) will be aseptically collected from the perimeter of each lagoon and composited. Samples will be collected by the researcher, unless it is deemed necessary to collect the samples using a single- or double-blind method. In either case, the samples will be transported to our laboratory in coolers, then stored at 5oC for no longer than 48 hours. Prior to DNA extraction, the composite samples will be thoroughly homogenized and 100-mL aliquots will be filtered through 0.2 'm filters. The filters will then be placed into bead beating tubes and processed to extract the DNA from the microbial community. The DNA will be amplified using universal primer sets (BA8F and UN1492R) and the subsequent PCR product will be cloned into pGEM-T Easy Vector. One hundred clones from each plate will be randomly selected, then grown in appropriate media. Afterwards, the plasmid from each clone will be isolated and sent to a commercial laboratory for sequencing. The 16S rDNA sequences will be identified using the BLAST database at the National Center for Biotechnology Information. In the subsequent year, samples will be collected from the same lagoons using the procedures described above. Based on the information collected during year 1, up to five bacterial pathogens will be selected for further study. The suspected targets are Campylobacter jejuni, Escherichia coli O157:H7, Salmonella, Clostridium perfringens, and Mycobacterium bovis. These pathogens will be quantified using standard culture-based methods and quantitative PCR. In addition to these bacterial pathogens, the following indicator organisms of fecal pollution will be cultivated: Escherichia coli, total coliforms, fecal streptococci, and somatic coliphage (bacterial virus).
Research conducted under this agreement contributes directly to Objective 2 of the in-house project by quantifying bacterial pathogens in dairy wastewater ponds. Methods to quantify bacterial pathogens in wastewaters were developed. Samples from 30 dairy lagoons were collected in the summer of 2011 for analysis. This information will be used to estimate the potential amount of pathogens that could be in wastewater used for irrigation. Information is exchanged with the cooperator on a regular basis through e-mail, phone calls, and meetings.