CICEET Progress Report for the period 3/15/05 Through 9/15/05

Project Title: F+ RNA Coliphages as Source Tracking Viral Indicators of Fecal Contamination
Principal Investigator(s): Mark D. Sobsey
Additional Investigator(s): Greg L. Lovelace
Project Start Date: 9/1/2003

Figures

Figure 1

Tables

Table 1

Table 2

Table 3

Table 4

Table 5

Tasks to meet objectives
Collection and analysis of water and shellfish samples: continue to perform analyses on those samples as they are delivered to the field laboratory; proceed with characterization of collected field isolates. Presentations of methods and partial study data at various meetings; production of workshop manual on methods

Progress on Tasks
Sampling at the NERR sites has concluded. The last site, at Apalachicola Bay NERR, was delayed due to complications caused by Hurricane Katrina on August 29 and re-sampling scheduled for early September was also canceled due to inclement weather in the Gulf Coast. Because of scheduling difficulties this Fall, the final Apalachicola Bay sample set cannot be performed As of this report, a total of 35 sample sets (out of a total of 36) have been collected from 9 of the 10 reserves that initially agreed to assist. One extra sample set has been collected from a separate local site to make up for the original site, which was dropped from the study.

Accomplishments
Sample collection is complete from all of the participating nine reserves. A total of 152 separate samples have been collected, consisting of 78 4-liter water samples, 34 pooled oyster samples, 28 pooled clam samples, and 12 pooled mussel samples. All collected samples have been processed by culture methods for bacterial indicators and for coliphages. All male-specific coliphage isolates have been RNase tested, and 95% of those 1156 isolates have undergone molecular genotyping for microbial source tracking. The remaining male-specific RNA coliphage isolates are in the Chapel Hill laboratory awaiting analysis. Somatic coliphage isolates (from each site and isolation medium) have been archived for further analysis when improved characterization methods are developed in the future.

The results based on data collected up to February 2005 were presented at the meeting of the Gulf and South Atlantic Shellfish Meeting, May 22-25, in Myrtle Beach, SC. On May 23d, Dr. Mark Sobsey and Greg Lovelace gave a talk entitled “New Indicator Methods For Virus Detection in Shellfish” to an audience of state shellfish sanitation program managers. The results were also presented as a poster entitled “Improved Methods for Detection of Coliphages in Coastal Water and Molluscan Shellfish” by Greg Lovelace at the American Society for Microbiology annual meeting in Atlanta, GA on June 8, 2005. That poster is currently available on the CICEET Project Explorer website. On July 11, 2005, at the invitation of Dr. Roger Fujioka, Greg Lovelace presented the talk “New Indicator Methods For Virus Detection in Coastal Water and Molluscan Shellfish” to an audience of students and faculty at the University of Hawaii in Honolulu. These improved methods for coliphage detection were presented at the International Workshop on Molluscan Shellfish Safety on August 12, 2005 in connection with the biannual meeting of the Interstate Shellfish Sanitation Conference in Point Clear, AL. Greg Lovelace and Brian Robinson of the NOAA Center for Coastal Environmental Health and Biomolecular Research conducted the workshop at the invitation of the USFDA Shellfish Laboratory in Dauphin Island, AL. A document entitled "Methods to Detect and Genotype Coliphage in Water and Shellfish" was produced for distribution at the workshop. It is available online.

Difficulties
One of the reserves, which originally agreed to participate, was unable to coordinate with the field laboratory and, as a result, was dropped from the study. Two more sets of sampling sites were identified on the Rachel Carson Component of the North Carolina NERR, which fit the study profile, and sampling was considered from these sites in order to compensate for the missing samples from the NERR that was dropped from the study. However, scheduling only allowed one set of samples from these alternative sites.

Molecular genotyping and source tracking of male specific coliphage was initially delayed for methods optimization and validation, although now most isolates have been characterized and are completed.

Project Objectives for Next Reporting Period

Objectives
Our plans for the remainder of the extended project call are to complete molecular characterization of the collected male-specific coliphage isolates, complete the final report; continue data presentations in scientific public forums (ie conferences, lectures, scientific journals, and posting to CICEET website and our own website.

Tasks to Meet Objectives
We have received a no-cost extension for this project through December 2005, and we hope to be able to complete the sample analysis and submit a final report by the end of 2005. To meet our objectives finish coliphage characterization, begin data analysis of the full data set which includes statistical analysis to test our research hypotheses; we will also begin writing sections of the final report; prepare our final report for publication in a peer-reviewed journal; develop material for presentations at conferences including the Estuary Research Federation (EFR) conference in the Fall of 2005.

Work Plan for Next Reporting Period
With sampling complete, statistical analysis of the data will take place, and a final report will be produced. Included in the final report will be an individual report for each participating NERR which will be shared with the Research Coordinators at each NERR. The final report will be produced by the end of 2005.

Anticipated Success in Meeting Project Objectives
We are confident that we will meet our objectives as we have done previously, and that the project will be completed by the end of Calendar year 2005.

Overall Project Timeline Update
As stated above, we have received a no-cost extension of the project through the end of the calendar year.

Preliminary Data
A total of 1156 male-specific coliphage isolates have been collected, of which 1005 were found to be RNA coliphages. 948 of those male-specific RNA coliphage isolates have been characterized as human or non-human type coliphages.

Coliphage are useful to identify fecally contaminated sites, to quantitatively portray impacts of fecal contamination at sites, and to track fecal contamination sources. A large number of coliphages, both somatic and male-specific, have been isolated and quantified in water and shellfish samples from the sites selected for this study. Coliphage are detected and quantified in most samples, with a range and prevalence of detection similar to bacterial indicators (see Table 3). Coliphages are prevalent at both fecally contaminated sites and sites classified as sufficiently clean to permit shellfishing (see Table 1, Table 2 and Table 3). Initial comparisons between bacterial indicators and viral (coliphage) indicators appear that coliphage may be an appropriate indicator of fecal contamination (see Table 5), but further statistical analysis of the data is needed to confirm this hypothesis.

When comparing coliphage detection methods, not all coliphage assays performed equally— the Two-Step Enrichment assay perform better than both the membrane filtration assay and Single Agar Layer assay at detecting coliphage in water, presumably because coliphage are at low concentrations in water (see Table 3). In shellfish, both coliphage methods were equivalent at detecting coliphage, because shellfish samples usually had high levels of coliphage if contaminated, making the detection limit of the assay less important for comparisons of presence/absence detection. (see Table 3)

Somatic and male-specific coliphages are present in water more frequently and at higher concentrations at all stations designated as “fecally contaminated” than at all stations designated as “uncontaminated” and this trend appears to dependent upon which method is used to recovery and detect coliphage (see Table 5). A graphical depiction of the analyses found in Table 5 is shown in Figure 1 as box-and-whisker plots of fecal indicator bacteria and coliphage concentrations. The box-and-whiskers plot highlights the range of concentrations of bacterial and viral indicators found at all contaminated and uncontaminated sites.

In characterizing F+ RNA isolates (see Table 4), mixtures of coliphage from human and non-human sources were found at both sites labeled as contaminated and uncontaminated, which needs to be further investigated by comparing sample sites and known sources of fecal contamination (possibly with GIS), and apply previously found effects of seasonality on coliphage distribution and survival. These added analyses will tease apart relationships between coliphage type and source of contamination to better understand the nature of coliphage for source tracking.

Dissemination
Dissemination of the collected data has been accomplished as required by CICEET in the form of progress reports. Research coordinators of the participating reserves have access to those reports from the Project Explorer at the CICEET website. In addition, as each set of samples is completed, a copy of the raw and tabulated data, in the form of an Excel spreadsheet file, is shared with the researcher in charge of sample collection at that site.

Conferences:
The research program and progress of the laboratory was presented at the US EPA BEACHES Program “National Beaches Conference” held October 13-15, 2004 in San Diego, CA. Greg Lovelace presented a paper entitled: “Male-Specific Coliphages as Indicators of Fecal Pollution in Coastal Recreational Waters.” That presentation included data from the on-going research of the current project.

The results based on data collected as of February 2005 were presented at the meeting of the Gulf and South Atlantic Shellfish Meeting, May 22-25, in Myrtle Beach, SC. On May 23d, Dr. Mark Sobsey and Greg Lovelace gave a talk entitled “New Indicator Methods For Virus Detection in Shellfish” to an audience of state shellfish sanitation program managers.

The results based on data collected as of February 2005 were presented as a poster entitled “Improved Methods for Detection of Coliphages in Coastal Water and Molluscan Shellfish” by Greg Lovelace at the American Society for Microbiology annual meeting in Atlanta, GA on June 8, 2005. That poster is currently available on the CICEET Project Explorer website.

On July 11, 2005, at the invitation of Dr. Roger Fujioka, Greg Lovelace presented the talk “New Indicator Methods For Virus Detection in Coastal Water and Molluscan Shellfish” to an audience of students and faculty at the University of Hawaii in Honolulu.

These improved methods for coliphage detection were presented at the International Workshop on Molluscan Shellfish Safety on August 12, 2005 in connection with the biannual meeting of the Interstate Shellfish Sanitation Conference in Point Clear, AL. Greg Lovelace and Brian Robinson of the NOAA Center for Coastal Environmental Health and Biomolecular Research conducted the workshop at the invitation of the USFDA Shellfish Laboratory in Dauphin Island, AL. A document entitled "Methods to Detect and Genotype Coliphage in Water and Shellfish" was produced for distribution at the workshop. It is available online.

Other dissemination events are planned and will continue through the duration of the project.

Expenditures
Expenditures are within the budget for this project.