ABSTRACT
This research developed a PCR method to identify swine fecal pollution in water, using a portion of the STII toxin gene from enterotoxigenic Escherichia coli as the target sequence. This method showed the gene to have a wide-spread geographical distribution and temporal stability; and the primers demonstrated high specificity, sensitivity, and reliability. A total of 110 DNA extracts from different animal fecal and human sewage samples were screened using the primers and no positives resulted. Centrifugation and filtration methods for concentrating E. coli seeded into stream, ocean, secondary effluent, and dairy lagoon waters resulted in detection limits at the femtogram and attogram levels. E. coli with the biomarker seeded into stream, ocean, and secondary effluent waters remained stable for approximately 2 weeks for all water types. Of the farm lagoon and waste samples tested, 94% were positive for the STII trait, regardless of the number of E. coli screened and 100% were positive when > or =35 E. coli isolates were screened. As the PCR product of the target sequence yielded a single band, the method is applicable to dot blot detection methodology, yielding great accuracy in determining the presence of swine fecal sources.
Subject(s)
Bacterial Toxins/genetics , Biomarkers , Enterotoxins/genetics , Escherichia coli/isolation & purification , Feces/microbiology , Swine/microbiology , Water Pollution , Animals , DNA Primers , Environmental Monitoring , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins , Humans , Polymerase Chain Reaction , Sensitivity and SpecificityABSTRACT
This research describes a method based on PCR to identify cattle fecal pollution in water using a portion of the heat labile toxin IIA (LTIIa) gene from enterotoxigenic Escherichia coli (ETEC). We describe the development of the primers and target. DNA extracts (221) from different animal fecal and human sewage samples were screened and showed no cross-reactivity. Minimum detection limits using centrifugation and filtration methods to concentrate E. coli seeded into stream, ocean, and secondary effluent waters were found to be at femtogram and attogram levels, respectively. Stability of the biomarker in stream, ocean, and secondary effluent waters was 2-4 weeks for all water types. Finally, 33 farm lagoon and waste samples were collected and 31 tested to validate the method; 93% were positive for the LTIIa trait when >1,000 E. coli were screened and 100% positive when >10(5) E. coli were screened. Prevalence of the toxin gene in the E. coli population affected the outcome of the analyses. The cow biomarker can be used in watershed studies to identify cattle waste with great accuracy if the appropriate numbers of E. coli are screened.
