MICROBIAL ECOLOGY AND MOLECULAR CHARACTERIZATION OF PATHOGENS IN FEEDYARD PLAYA AIR AND WATER SAMPLES IN THE HIGH PLAINS OF TEXAS

Authors: Rice, William; Purdy, Charles

Submitted to: American Society of Microbiologists Abstracts
Publication Type: Abstract Only
Publication Acceptance Date: 10/16/2002
Publication Date: 11/7/2002
Citation: Rice, W.C., Purdy, C.W. Microbial Ecology and Molecular Characterization of Pathogens in Feedyard Playa Air and Water Samples in The High Plains of Texas. American Society of Microbiologists. 2002. Abstract p. 22.

Interpretive Summary: Concentrated animal feeding operations (CAFO) may impact the environment due to nutrient inputs present in the manure, and the sheer volume of manure that must be removed. Consequently, management issues such as odor, presence of pathogens, endotoxins, and dust emissions are introduced. The impact of these nutrient inputs and pathogens on the microbial ecology of feedyard air and retention pond water is under investigation. A key objective of this project is to develop the tools and necessary database information to: 1) rapidly detect environmental E. coli O157:H7 isolates, 2) assess the biodiversity of environmental Escherichia sp. inhabiting feedyard retention ponds, and 3) to monitor the fate and transport of E. coli O157:H7 in the environment over time. Airborne microbial concentrations of 7 CAFO were determined by using 2- and 6-stage Andersen biological cascade impactors and SKC air samplers positioned upwind and downwind of each feedyard retention pond while water samples were obtained from each feedyard retention pond. All samples collected were cultured on a variety of nonselective and selective medias. Water samples were extracted to obtain bulk nucleic acids using MO BIO water kits and selectively enriched for E coli O157H:7 or Salmonella sp. Molecular methods (AP-PCR) were developed to detect and discriminate among various E coli sp by taking advantage of the fact that the complete genomes of E. coli strains: K12-MG1655, O157:H7-EDL933 and O157:H7B-Sakai have been sequenced. A global alignment of all three genomes (approximately the 1st 0.5 megabases) was conducted using the Clustal W program. Genomic regions of high homology interspersed with insertions/deletions were selected for the design of primers to be used in AP-PCR assays. Seven PCR primer pairs (A-G) were selected from several hundred possible pairs that were predicted to generate a single diagnostic amplicon (250 to 1800 bps) per MG1655, EDL-933, or Sakai genomes. As predicted, diagnostic amplicons were observed in PCR reactions using genomic DNA obtained from MG1655, EDL-933, and Sakai strains. The usefulness of these primer pairs was validated employing the DEC A set of strains (n=72) obtained from the National Food Safety and Toxicology Center, Michigan State University and suspected E coli O157:H7 isolates obtained from enrichments of feedyard retention pond samples. RAPD-PCR studies using 10-mer primers from Operon were also identified that were capable of generating useful DNA fingerprints of environmental isolates of E coli O157:H7 in order to assess the biodiversity of E. coli strains inhabiting feedyard retention ponds.

Technical Abstract: Concentrated animal feeding operations (CAFO) may impact the environment due to nutrient inputs present in the manure, and the sheer volume of manure that must be removed. Consequently, management issues such as odor, presence of pathogens, endotoxins, and dust emissions are introduced. The impact of these nutrient inputs and pathogens on the microbial ecology of feedyard air and retention pond water is under investigation. A key objective of this project is to develop the tools and necessary database information to: 1) rapidly detect environmental E. coli O157:H7 isolates, 2) assess the biodiversity of environmental Escherichia sp. inhabiting feedyard retention ponds, and 3) to monitor the fate and transport of E. coli O157:H7 in the environment over time. Airborne microbial concentrations of 7 CAFO were determined by using 2- and 6-stage Andersen biological cascade impactors and SKC air samplers positioned upwind and downwind of each feedyard retention pond while water samples were obtained from each feedyard retention pond. All samples collected were cultured on a variety of nonselective and selective medias. Water samples were extracted to obtain bulk nucleic acids using MO BIO water kits and selectively enriched for E coli O157H:7 or Salmonella sp. Molecular methods (AP-PCR) were developed to detect and discriminate among various E coli sp by taking advantage of the fact that the complete genomes of E. coli strains: K12-MG1655, O157:H7-EDL933 and O157:H7B-Sakai have been sequenced. A global alignment of all three genomes (approximately the 1st 0.5 megabases) was conducted using the Clustal W program. Genomic regions of high homology interspersed with insertions/deletions were selected for the design of primers to be used in AP-PCR assays. Seven PCR primer pairs (A-G) were selected from several hundred possible pairs that were predicted to generate a single diagnostic amplicon (250 to 1800 bps) per MG1655, EDL-933, or Sakai genomes. As predicted, diagnostic amplicons were observed in PCR reactions using genomic DNA obtained from MG1655, EDL-933, and Sakai strains. The usefulness of these primer pairs was validated employing the DEC A set of strains (n=72) obtained from the National Food Safety and Toxicology Center, Michigan State University and suspected E coli O157:H7 isolates obtained from enrichments of feedyard retention pond samples. RAPD-PCR studies using 10-mer primers from Operon were also identified that were capable of generating useful DNA fingerprints of environmental isolates of E coli O157:H7 in order to assess the biodiversity of E. coli strains inhabiting feedyard retention ponds.