CICEET Progress Report for the period 3/01/04 through 9/01/04

Project Title: Integration of Ribotyping into Existing Monitoring Programs to Identify Fecal Contamination Sources in GBNERR
Principal Investigator(s): Stephen H. Jones

Accomplishments
Scheduled Tasks:
The project is complete and the final project report writing is in progress. Below is an updates report on project tasks. As instructed, no data are included.

Progress on Tasks
Plan sampling schedules/coordinate with other agency personnel
There were a number of different related projects ongoing during this project where coordination of sampling has been helpful to all projects. There have been numerous meetings and discussions held with various state agency personnel involved in the other projects to discuss how best to cover all projects’ sampling needs in the most efficient manner. Some adjustments were made in several related projects and samples from those projects constitute the majority of samples collected as part of this CICEET project. The other projects involved include the NH National Coastal Assessment (NHNCA) monthly water quality monitoring, the GBNERR SWMP monthly monitoring and the NH Gulfwatch program. The benefit to this CICEET project is that other data collected as part of these programs will enhance the ability to interpret environmental conditions associated with the source species identifications.

Conduct water and source sampling, modify approach
Water sampling was initiated in Fall, 2001 to see what levels of Escherichia coli were present at the sampling sites under different conditions. Isolates have been saved for ribotyping since April, 2002. These samples include monthly samples collected as part of the SWMP program (4 sites; low and high tide), monthly samples collected as part of the NHNCA program (13 sites; low tide), monthly samples collected along a transect from Oyster River throught Little and great bays and into the Squamscott River (7 sites; low tide) and annual sampling for the NH Gulfwatch program (6 sites/y; water and shellfish: blue mussels and oysters). Water quality data collected as part of the other programs for the samples used for ribotyping have been compiled.

Source sampling has been underway since November, 2001. Potential source species include those suspected of being significant sources in the study areas, particularly humans/septic systems, WWTFs, buffaloes, cows, migratory birds, deer and other wildlife. E. coli isolates from these source species have been identified and processed for ribotyping.

Present findings to interested state and regional agencies
The results of this and other related ribotyping studies conducted in coastal NH have been made at many different scientific, general public, agency and other types of meetings. In most instances a PowerPoint presentation was made or a poster was presented. Presentations have been made to local, regional, national and international (Brazil, Scotland) meetings during the last reporting period.

Difficulties Encountered
The project has run with no major difficulties. One significant development that has had a large impact on this study is the purchase of a RiboPrinter by our lab during the summer of 2002. We did not run any project isolates through our established manual ribotyping procedures in anticipation of being able to use the RiboPrinter for analyzing all project-related isolates. All source species for coastal New Hampshire and the region (ME, MA, VT) have now been re-run with the RiboPrinter. The total number of isolates in the database used for analysis of water samples was 1050, including 775 from NH that have been used as a local, geographically-specific database for initial analysis of isolate sources. This process of analyzing water samples has been completed for samples collected through November, 2003, including the additional samples collected to complete the final sampling needs for one of the monitoring programs.

The final analyses were conducted during March-May, 2004. The initial results using only EcoR1 enzyme digestion gave numerous similar ribopatterns for both water sample and source species E. coli isolates. This made it impossible to identify the source species for some samples. The final set of analyses being conducted used a second restriction enzyme, PvuII, on a subset of the source species database and water samples from one of the sampling programs. These analyses are now completed.

Anticipated Success in Meeting Project Objectives in Scheduled Project Period
The project is complete and the final project report writing is in progress.

Preliminary Data
For the NCA samples, samples containing >40 E. coli/100 ml were chosen for ribotyping, although not all samples with those concentrations had available isolates. This cut off value provided isolates from a set of samples that serve as a baseline for temporal and spatial source identification at the sites with the highest E. coli concentrations. For the SWMP program, the criteria for choosing samples for ribotyping was slightly different. Samples with a range of E. coli concentrations were chosen to provide some representative analysis from all sites. Relatively low concentration were chosen for CML and AP because they had consistently low E. coli concentrations. Because the SWMP program provides isolates at both low and high tides, isolates were analyzed for some dates at both tidal stages. E. coli isolates from the Gulfwatch and project-specific samples represented all samples collected.

The E. coli analysis results for the project-specific monitoring, along the Oyster River-Squamscott River transect, showed the expected higher concentrations in the rivers and lower levels in Little and Great bays.

The analysis for ribotype identification of the project-specific sites in Great Bay and the Oyster River and the NH Gulfwatch Program have been completed for the Fall, 2002 samples. The results showed humans, birds and wildlife to be somewhat equally significant for the Great Bay samples, and more significant than pets and livestock, for the combined database for the 7 sample sites. Livestock were also significant for the Gulfwatch samples, and pets were not as significant once again.

The types of source species identified for isolates from the SWMP samples showed humans to be the most significantt source type, with wild animals and livestock of lesser significance, and only low levels of pets and birds. For the NCA isolates, wild animals were the most significant source species type, followed by humans and livestock, with only low levels of pets and birds. Thus, some consistent results emerged from these studies for the Great Bay Estuary and an approach for utilizing ribotyping in monitoring programs is also becoming clearer. Data for individual species and sites provide more detailed information that will be useful for using these results to improve water quality.

More recent work has been directed at source species identification in oysters and overlying waters at two sites in Great Bay Estuary. The Oyster River site was more highly contaminated than the Nannie Island site and shellfishing is prohibited. The waters around Nannie Island are classified as approved for shellfishing. Both sites are also being sampled as part of a new CICEET project that began in October, 2003. The results for samples collected from May-October, 2003 showed livestock species to be most significant in both tissue and water at both sites, especially at Nannie Island. Humans were next most significant at both sites, especially at the Oyster River site, while pets, birds and wild animals were generally present at low levels. There were some differences between sites and between tissue and water samples.

A second enzyme digestion of DNA using PvuII enabled identification of more isolates because it could differentiate between isolate DNA banding patterns that had been identical using only EcoR1 digestion. Isolates from known source species were digested with PvuII to construct a small database for identification of water and oyster sample isolates collected from the Oyster River and Nannie Island. Because of the expense for doing this, we focused on isolate EcoR1 patterns that had either 8 or 9 DNA bands. We chose those 8-9 band patterns that were common amongst different source species to see if a second enzyme digestion could differentiate between them. The same approach was used for water samples in that isolates chosen for PvuII digestion were those that matched more than one source species and had 8 or 9 DNA bands.

We found that digestion of isolate DNA with PvuII gave identical patterns for 5 isolates from one individual human, confirming what EcoR1 digestion had shown that these appear to be clones. PvuII digestion of 8 isolates from a cat that had given identical patterns using EcoR1 gave 8 distinct yet closely related patterns, showing that these were not clones. Digestion of DNA from isolates collected from different source species that had given identical patterns using EcoR1 gave different patterns for some and identical patterns for others. All the PvuII patterns produced from known isolates (191) were compiled into a database for comparison with water and oyster sample patterns also digested with PvuII. The increased number of isolates for which source species were identified enabled a better understanding of sources to oysters and water at the two study sites.

Tasks and activities for the next reporting period

Tasks for the next reporting period
The final report will be finished. The final report will include a full analysis of the data, its interpretation, and how ribotyping can best be integrated into estuarine water quality monitoring programs.

Work plan to accomplish tasks
Write final report     December, 2004

Concerns or difficulties
None.

Expenditures
Records are maintained and accounts managed by the COLSA BSC at Rudman Hall.