Improved Methods for Fecal Pathogen Detection Monitoring and Management

Non-Technical Summary: The freshwater, estuarine, and nearshore marine waters of California provide critical resources for drinking water, food production, industry, recreation, and wildlife habitat. Contaminated water compromises the quality of these resources. Fecal pollution is a significant and insidious threat to human and animal health. Potential sources of fecal pollution include agricultural runoff containing livestock feces, sewage outfalls, and stormwater runoff. The practice of intensive food animal production has also come under increasing attack as a major source of water pollution. As the top dairy state, California is the nation's number one dairy producer of both milk and manure, with approximately 30 million tons of manure being produced by dairy cattle each year in. Management of this fecal waste as well as the protection of our watersheds as a source of both recreational and drinking water, are two of the state's most pressing environmental issues. Water quality monitoring has historically focused on heavy metals, anthropogenic chemicals, and bacterial coliform counts. This methodology does not address the risk due to other pathogenic organisms, such as protozoal parasites that may be present in the aquatic environment as a result of fecal contamination. Cryptosporidium, Giardia, and Toxoplasma are well-known protozoal causes of disease in people and animals, and are usually spread by water contaminated with human and/or animal feces. Similarly, the risk of exposure to pathogenic bacteria is not taken into consideration in water quality monitoring despite the fact that waterborne exposure to some species of bacteria, most notably Vibrio cholerae, Campylobacter, and Salmonella, are known to cause severe diarrheal disease in humans. Our efforts to develop methods for improved T. gondii oocyst concentration and detection will have immediate practical applications for large-scale testing of California surface waters, bivalves, sewage outfalls, and feces from domestic and wild felids. The direct outcome benefit of this component of the project is the production of a sensitive, specific and practical method for the concentration and detection of T. gondii oocysts which will be validated for testing water in coastal areas and coastal-draining watersheds. Overall, federal and state agencies charged with water quality and species management, including range expansion issues, will benefit from the pathogen risk-exposure assessment methods and data produced by this project. Evaluation of the sources of fecal contamination and development of mitigation strategies will help to reduce pathogen pollution in the freshwater and estuarine components of the watershed which provides water to the near-shore marine environment. All these components comprise a vital ecosystem and resource for humans and animals. This project has important implications for the management of agricultural wastewater, as well as threatened wildlife species, and furthers our understanding of the human health risks of local shellfish consumption and potential marketability of shellfish obtained from waters exposed to surface runoff or sewage. <P> Approach: Hypothesis 1: Plasmids will be constructed containing the entire DNA coding sequence for outer wall proteins of T. gondii published on the Toxoplasma genome database. Each described protein will be will be used for DNA immunizations. The target gene sequence will be PCR amplified using specific primers with overlapping start and stop codons. Recombinant proteins, representing each of the seven identified outer wall proteins, will be produced following established protocols. Seronegative BALB/c mice will be immunized with each plasmid construct and each recombinant protein. After immunization mice will be serologically screened. Mice demonstrating high titers to oocyst wall antigens will be sacrificed and their spleen cells will be harvested for hybridoma formation. Monoclonal antibody production will follow standard methodology for spleen cell/myeloma fusion. Hybridomas will be screened by testing supernatant for reactivity to oocyst wall preparations by ELISA and IFA using intact oocysts. Antibody isotype will be determined and hybridomas producing high concentrations of IgG isotype will be selected, cloned, and cultures expanded and supernatant will be collected for monoclonal antibody purification. Monoclonal antibodies will be conjugated to paramagnetic beads for IMS. T. gondii oocysts will be visually identified following IMS concentration in water samples by imunofluorescence assay (DFA). The sensitivity of the monoclonal antibodies for IMS and DFA will be determined by spiking experiments with a variety of water types. The specificity of the monoclonal antibodies will be evaluated by testing against closely related organisms. Hypothesis 2: Wastewater samples will be collected from cooperating livestock operations in four regions of the USA. The water samples will be concentrated down to volumes of 100-200 mls using ultrafiltration. Cryptosporidium and Giardia spp will then be further concentrated and quantified from samples using EPA approved Immunomagnetic Separation (IMS) and DFA techniques. Protozoa-positive slides will then be scraped to recover the protozoal DNA and molecular genotyping will be done to characterize human vs animal strains. Standard E. coli FIB testing and bacterial culture will be performed at UCD or at local laboratories to meet holding time requirements. Bacteroidales host-specific assays will also be performed to evaluate the contribution of cow, dog and human fecal markers. Statistical analyses will utilize a variety of software including Stata, R, Minitab, and Mathematica to evaluate and assess trends in the data within individual studies as well as for comparisons across studies in meta-analyses. Additional modeling approaches will be developed specifically for Bacteroidales and QMRA frameworks. The most promising QMRA tools will be further evaluated and compared for their abilities to characterize risk pertaining to specific pathogens as well as contributing host sources. The predicted host-contribution results will be compared with the known presence of the host groups at the various study sites where samples of wastewater and ambient waters were collected.