CICEET Progress Report for the period 9/15/04 Through 3/15/05

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
Continue to schedule and coordinate collections at participating NERRs, supplying shipping materials as needed; continue to perform analyses on those samples as they are delivered to the field laboratory; proceed with characterization of collected field isolates

Progress on Tasks
Sampling at the NERRS sites has continued at an acceptable pace. As of this report, a total of 28 sample sets (80% of the planned total) 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
As of this date, sample collection is complete from 4 of the participating 9 reserves, with spring samples remaining for 4 reserves and summer samples remaining for 3 reserves. All of these remaining sample sets have been scheduled through the end of August, 2005.

A total of 126 separate samples have been collected, consisting of 64 water samples, 30 oyster samples, 24 clam samples, and 8 mussel samples. All collected samples have been processed for bacterial indicators and for coliphages through the RNAse testing phase. Several sets of somatic coliphage isolates, some from each site and isolation medium collected so far, have been archived for further analysis when improved characterization methods become developed in the future. A total of 1081 male-specific coliphage isolates have been collected, of which 790 were found to be RNA coliphages. 343 of those male-specific RNA coliphage isolates have been characterized as human or non-human type coliphages. The remaining male-specific RNA coliphage isolates have been shipped to the campus laboratory in Chapel Hill and are awaiting molecular genotyping tests for source characterization

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 samples will be collected from these sites as time allows to in order to compensate for the missing samples from the NERR that was dropped from the study.

Methodological problems in the Chapel Hill laboratory significantly delayed the beginning of the molecular genotyping and source characterization of the collected male-specific RNA coliphage isolates. However, as detailed above, the first of those characterizations have been completed, and the remaining isolates collected to date have been shipped to the Chapel Hill laboratory and are being characterized as of this date.

The 2004 Hurricane Season proved disastrous to our efforts toward developing a field coliphage test kit. The doctoral student who was working to help develop that kit had relocated her effort from the laboratory in Chapel Hill to the US EPA Laboratory facility in Gulf Breeze, Florida, which suffered a hit from Hurricane Ivan. As a result, all of the student’s work was destroyed, and our test kit work was halted. To date, we have been unable to resume work on the test kit, and we regret that we cannot recover from the devastation of the hurricane. However, we have submitted a proposal to CICEET for the next funding cycle which does include plans for the development of an improved rapid-detection rapid-characterization field coliphage test kit, which will be a significant improvement on our original idea.

It had been part of our original plan to have Susan Lovelace, who was at the time the Education Coordinator for the North Carolina National Estuarine Research Reserve, assist our laboratory in the development of educational materials and workshops for the dissemination of the methods and test kits to coastal decision-makers and other interested parties. However, Ms. Lovelace is no longer with the NC NERR and is now the education coordinator for the NOAA Oceans and Human Health initiative, working out of Hollings Marine Laboratory in Charleston, SC. As a result, we have no current plan for the outreach segment of this study.

Project Objectives for Next Reporting Period

Objectives
According to the timeline, the final six months of the study will be devoted to the completion of the sampling of designated sites at the NERRs. Also included in the timeline are reporting of the initial findings and tools, the production of dissemination products, user workshops, and the preparation of the final report.

Tasks to Meet Objectives
As stated above, we regret that we will not be able to produce the field test kit for coliphages, and so we will not be conducting workshops to demonstrate and teach others to use those kits or the methods for coliphage detection by the end of the study period in September 2005. However, we have applied to CICEET for 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.

Work Plan for Next Reporting Period
Continue to coordinate collections at participating NERRs, supplying shipping materials as needed; continue to perform analyses on those samples as they are delivered to the field laboratory and complete the field sampling and initial analyses by the end of August 2005. Continue the molecular genotyping and characterization of collected male-specific coliphage isolates. Produce a final report by the end of 2005.

Anticipated Success in Meeting Project Objectives
We are confident that the project will be completed by the end of Calendar year 2005.

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

Preliminary Data
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. It appears that these coliphages are quite prevalent, at least at fecally contaminated sites and perhaps at many of those sites classified as sufficiently clean to permit shellfishing. (See Table 1, Table 2 and Table 3). These detected coliphages may be useful to identify fecally contaminated stations and sites, to quantitatively portray impacts of fecal contamination at sites and stations and to track fecal contamination sources. Characterization to date of some of the collected F+ RNA isolates (See Table 4) has shown a large proportion (329 out of 352) of coliphages from non-human sources at sites characterized as both contaminated and uncontaminated. Of particular interest is the presence of coliphages from human sources at sites characterized as uncontaminated (highlighted in red in the table).

Preliminary analysis of current data (See Table 5) indicate that somatic and male-specific coliphages are present in water more frequently and at higher concentrations at stations designated as “fecally contaminated” than at stations designated as “uncontaminated” by NERRS managers. The results of these analyses to date are shown in Figure as box-and-whisker plots of fecal indicator bacteria and coliphage concentrations.

Further analysis and characterization studies on F+ coliphage isolates are now in progress. All male-specific coliphage isolates collected to date have been tested for susceptibility to RNAse and characterized as DNA, RNA, or a mixture. These F+ coliphage isolates are awaiting further molecular tests in an effort use their group or type identification to determine their source. Several sets of somatic coliphage isolates, some from each site and isolation medium collected so far, have been archived for further analysis when the characterization methods are developed in the future.

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.

An abstract entitled “Male-Specific Coliphages as Indicators of Fecal Pollution in Coastal Recreational Waters” has been submitted to the American Society of Microbiologists for inclusion as a poster presentation of the study data and preliminary conclusions at the Society’s annual meeting in June, 2005.

An abstract has been submitted to the Estuarine Research Foundation for inclusion as an oral presentation of the study data and preliminary conclusions at the Foundation’s annual meeting in October 2005.

Expenditures
Expenditures are within the budget for this project.