Grantee Research Project Results
2000 Progress Report: The Development of a DNA Based Specific Assay for Pfiesteria piscicida in Water and Sediments
EPA Grant Number: R827084Title: The Development of a DNA Based Specific Assay for Pfiesteria piscicida in Water and Sediments
Investigators: Oldach, David , Rublee, Parke
Institution: University of Maryland - Baltimore , University of North Carolina at Greensboro
EPA Project Officer: Packard, Benjamin H
Project Period: October 8, 1998 through October 7, 2001 (Extended to August 31, 2002)
Project Period Covered by this Report: October 8, 1999 through October 7, 2000
Project Amount: $709,537
RFA: Ecology and Oceanography of Harmful Algal Blooms (1998) RFA Text | Recipients Lists
Research Category: Water Quality , Harmful Algal Blooms , Water , Aquatic Ecosystems
Objective:
The association of Pfiesteria-like dinoflagellates with fish kill events and adverse human health effects has highlighted the need for research aimed at predicting, mitigating, and preventing such occurrences. However, the unique life cycle of Pfiesteria-like dinoflagellates, the absence of axenic culture for their study, the biohazard associated with attempted culture of toxin-producing organisms, and the laborious methods of scanning electron microscopy for morphological characterization have hampered progress in these endeavors. In Phase 1 of the proposed project, the investigators developed a molecular approach utilizing dinoflagellate-specific polymerase chain reaction (PCR) primers (18S rRNA), and the heteroduplex mobility assay (HMA) to identify characterized and novel dinoflagellate gene sequences (specifically Pfiesteria piscicida and Pfiesteria shumwayae) within cultures and environmental samples (Oldach et al. 2000). Our laboratories then began collaborations with other laboratories to determine clonality of dinoflagellate cultures and we also began to work with state agencies to build a database of environmental parameters related to presence or absence of Pfiesteria species as a result of comprehensive monitoring programs.The objectives for Phase II of the project included: (1) utilization of PCR and heteroduplex mobility based assays (in combination with sequencing and phylogenetic analyses) to determine clonality, distribution and novel sequences of Pfiesteria-like dinoflagellates. This work is done in collaboration with investigators at laboratories isolating and culturing dinoflagellates from varying geographical locations; (2) SSU sequence determination of Pfiesteria-like isolates to expand our dinoflagellate sequence matrix; (3) deployment of a real-time quantitative assay for the presence of Pfiesteria piscicida and Pfiesteria shumwayae in environmental samples collected by state agencies from New York to Texas as part of estuarine monitoring programs; (4) correlation of presence/absence of Pfiesteria species in the water column with reports of human health effects from an ongoing cohort study and with physicochemical and biological data collected in conjunction with water samples; and (5) development of a method for extraction of dinoflagellate DNA from sediment to be used in combination with the real-time PCR-based assays to screen for Pfiesteria species cysts. These investigations are being undertaken in order to fully understand the organisms' ecology and distribution (given the observation that detection of the organism in the water column itself is episodic, and, sometimes ?ephemeral' even in association with recent fish kill events).
Progress Summary:
At the beginning of this project period we developed real-time PCR-based assays for the detection of Pfiesteria piscicida and Pfiesteria shumwayae and rigorously tested them for specificity and sensitivity (Bowers et al. 2000). We routinely employed these molecular techniques in experiments with collaborating laboratories conducting studies with Pfiesteria and Pfiesteria-like species to confirm culture clonality, to develop sequence information for novel species, and to determine presence/absence of Pfiesteria species in cultures, environmental samples and fish kill bioassay experiments. Ongoing confirmation studies between molecular laboratories (D. Oldach; P. Rublee; K. Reece, Virginia Institute of Marine Science) have been developed to compare methods, specificity and sensitivity.During this period we were able to devise a method for extraction of dinoflagellate DNA from sediment. This DNA is PCR-ready and may be used with our real-time Pfiesteria-specific assays. This has also allowed us to incorporate sediment sampling as part of our collaborative efforts with state agencies during routine sampling and as part of event response sampling, thus expanding and enhancing our collaborations with other laboratories. Based on preliminary results utilizing this method, we (Oldach lab) have begun working on a sediment core project (funded by Maryland Sea Grant) with The Johns Hopkins University (PI: Grace Brush). We will be analyzing dinoflagellate DNA from dated strata of sediment cores. Dating will be performed by Dr. Brush, and will be based on a variety of sediment core parameters. The cores will be obtained from three locations to look for presence or absence of Pfiesteria-species DNA and to determine the structure of the dinoflagellate community, as reflected by cyst DNA, at different time points in (geologically recent) history. These investigations may allow us to draw inferences regarding relationships between the distribution and abundance of Pfiesteria-species and environmental transformations occurring in these waters over the past 500 years.
During this report period, our continuing collaboration with North Carolina State University (J. Burkholder) has involved screening of hundreds of cultures derived from environmental isolates and fish kill bioassays to determine presence/absence of Pfiesteria species. HMA has been used to determine clonality of cultures for these experiments, and 18S sequencing and phylogenetic analyses have been utilized for comparison of various strains of Pfiesteria and Pfiesteria-like species present in toxic and non-toxic samples derived from fish kill bioassays. We have used the same molecular approach in our collaboration with the Bigelow Laboratory for Ocean Sciences (CCMP; R. Andersen) to aid in characterization of Pfiesteria species isolated from various geographical regions. HMA analyses and sequence data for over 30 different isolates has been combined with morphological data to more fully characterize these species. This information has been made available to other researchers via the National Center for Culture of Marine Phytoplankton (CCMP) Web Site. Ongoing Pfiesteria species screening and 18S sequencing is also being employed in our collaboration with Old Dominion University (PI: Harold Marshall) to fully characterize Pfiesteria-like species being used in fish bioassays and to identify novel species, thus further expanding our dinoflagellate sequence matrix. To date, approximately 80 isolates have been sequenced and these data will be used to complement morphological data obtained through scanning electron microscopy. International collaboration has continued with the University of Oslo, Norway (K. Jakobsen; D. Klaveness) to explore whether Pfiesteria species may be found in other parts of the world. More than 30 Pfiesteria-like isolates derived from sediment collected along Norway's shoreline were screened with our Pfiesteria-specific real-time assays. Through screening and 18S sequencing, two cultures were confirmed as novel strains of Pfiesteria piscicida and further characterization of these cultures is in progress. International collaboration has also been established with researchers in 15 other countries, who have provided samples for analysis from estuarine locations. Pfiesteria shumwayae has been positively identified in samples from New Zealand, and P. piscicida has been found in coastal waters of Latvia. International collaborations are continuing with these and other countries.
During this project period, we have maintained relationships with state agencies (Maryland Department of Natural Resources; Delaware Department of Natural Resources and Environmental Control, Suffolk County [New York] Department of Health, New York Department of Environmental Conservation, New Jersey Department of Environmental Protection, Virginia Department of Environmental Quality, South Carolina Departments of Health and Natural Resources, Georgia Department of Natural Resources, University of Texas and Texas Department of Parks and Wildlife, Florida Department of Environmental Protection) interested in monitoring for Pfiesteria species and correlating that data with ecological parameters. Approximately 2,300 environmental estuarine samples (including 300 sediment samples) were screened for the presence of P. piscicida (~5 percent positive) and P. shumwayae (~3 percent positive) during this report period. Sediment samples have shown a higher incidence of positive assays than water samples. The results have also extended the southern limit of the distribution of Pfiesteria sp. to the southern tip of Texas. This distribution data is being analyzed in combination with physical parameters to determine temporal and spatial distribution of these two organisms. Currently this is being done most intensively in the Chesapeake Bay with the Maryland Department of Natural resources. Monitoring approaches and partial analyses have been presented in oral and poster form at several conferences. Similar analyses are also being performed for samples collected in Delaware's inland bays, where 150 water samples were analyzed for P. piscicida (9 percent positive) and P. shumwayae (2 percent positive).
In addition to routine screening of environmental water samples collected by these agencies, we have been involved in several small projects with various other agencies working with Pfiesteria species. During the summer of 2000, we assayed samples collected by Horn Point Environmental Laboratory (University of Maryland Center for Environmental Science) from transects on the Pocomoke (4) and Chicamacomico (1) Rivers and from a diel study performed on the Pocomoke. These studies were designed to look at distribution in these two previously affected rivers and to determine diel migration of the organisms in the water column. P. piscicida was not detected in any of these samples, while P. shumwayae was detected in one. In August and September, we collaborated with Maryland Department of the Environment (R. Kroll) in screening approximately 100 samples for Pfiesteria species. These samples were collected pre- and post-storm event on the Pocomoke as part of a study to determine effects of trace metals in the water column. In September, we also collaborated with the United States Army (T. Shedd) in their study of fish behavior and response to environmental conditions on the Chicamacomico River.
Our molecular approach has been used to develop real-time PCR assays for other harmful algal species, including Gymnodinium breve and Gymnodinium galatheanum (Tengs, et al., in press). In the development of the G. breve assay, we have strengthened our collaborative relationship with Florida Marine Institute (PI: Karen Steidinger). Screening of more than 20 cultures of Pfiesteria-likes and G. breve received from FMI has been coupled with 18S sequencing in support of morphological characterizations.
Future Activities:
Our laboratories will continue to assay environmental water and sediment samples for Pfiesteria piscicida and Pfiesteria shumwayae. In particular, intensive collaboration with Maryland DNR will continue in 2001. Water samples will be collected during the winter months to detect the fate of the organisms in the water column during sub-optimal conditions, but the bulk of the samples will be taken from April through October (>100 samples per month) as part of their comprehensive monitoring program. In addition, we will assay for these organisms from sediment being collected from 12 sites on 12 different tributaries throughout the Chesapeake Bay. Results from both water and sediment samples will be used to correlate the presence or absence of Pfiesteria species with possible adverse fish and human health effects and ecological parameters leading to a better understanding of temporal and spatial distribution.Experiments are currently underway in our laboratories to extensively study the effects on detection limits of both real-time PCR assays of high, moderate and low background DNA encountered in environmental samples. Based on these results, an appropriate standard curve will be developed and utilized on archived samples from 2000 and concentrations of P. piscicida will be determined and reported with any positive samples during the 2001 sampling season. Current experiments are also underway to multiplex the PCR, which combines both the P. piscicida and P. shumwayae assays in one reaction, thereby reducing assay time and cost.
We also will continue our collaborations with laboratories interested in isolation of Pfiesteria-species from various geographical locations. This will result in increased knowledge of distribution, discovery of novel strains, an increase in the number of facilities maintaining Pfiesteria cultures, and build on our dinoflagellate sequence matrix. This matrix will be used in experiments employing molecular techniques (HMA, 18S sequencing and dinoflagellate-specific PCR) to assess the dinoflagellate community in water and sediment samples from various locations.
Journal Articles on this Report : 5 Displayed | Download in RIS Format
Other project views: | All 90 publications | 23 publications in selected types | All 17 journal articles |
---|
Type | Citation | ||
---|---|---|---|
|
Bowers HA, Tengs T, Glasgow Jr HB, Burkholder JM, Rublee PA, Oldach DW. Development of real-time PCR assays for rapid detection of Pfiesteria piscicida and related dinoflagellates. Applied and Environmental Microbiology 2000;66(11):4641-4648. |
R827084 (2000) R827084 (2001) R827084 (Final) |
Exit Exit Exit |
|
Oldach DW, Delwiche CF, Jakobsen KS, Tengs T, Brown EG, Kempton JW, Schaefer EF, Bowers HA, Glasgow Jr HB, Burkholder JM, Steidinger KA, Rublee PA. Heteroduplex mobility assay-guided sequence discovery: elucidation of the small subunit (18S) rDNA sequences of Pfiesteria piscicida and related dinoflagellates from complex algal culture and environmental sample DNA pools. Proceedings of the National Academy of Sciences of the United States of America 2000;97(8):4303-4308. |
R827084 (2000) R827084 (2001) R827084 (Final) R825551 (Final) |
|
|
Rublee PA, Kempton J, Schaefer E, Burkholder JM, Glasgow Jr HB, Oldach D. PCR and FISH detection extends the range of Pfiesteria piscicida in estuarine waters. Virginia Journal of Science 1999;50(4):325-335. |
R827084 (2000) R827084 (2001) R827084 (Final) R825551 (Final) |
Exit |
|
Rublee PA, Kempton JW, Schaefer EF, Allen C, Harris J, Oldach DW, Bowers H, Tengs T, Burkholder JM, Glasgow Jr HB. Use of molecular probes to assess geographic distribution of Pfiesteria species. Environmental Health Perspectives 2001;109(Suppl 5):765-767. |
R827084 (2000) R827084 (2001) R827084 (Final) R825551 (Final) |
|
|
Tengs T, Bowers HA, Ziman AP, Stoecker DK, Oldach DW. Genetic polymorphism in Gymnodinium galatheanum chloroplast DNA sequences and development of a molecular detection assay. Molecular Ecology 2001;10(2):515-523. |
R827084 (2000) R827084 (Final) |
Exit Exit |
Supplemental Keywords:
estuary, fish health, human health, Atlantic coast., RFA, Scientific Discipline, Geographic Area, Water, Waste, Ecosystem Protection/Environmental Exposure & Risk, Contaminated Sediments, State, Oceanography, Environmental Microbiology, algal blooms, Ecological Risk Assessment, Ecology and Ecosystems, marine ecosystem, bloom dynamics, dinoflagellates, DNA based molecular diagnostics, fish kills, contaminated sediment, phytoplankton, polymerase chain reaction, Maryland (MD), pfiesteria, South Carolina (SC), North Carolina (NC), ECOHAB, gene sequencesRelevant Websites:
http://www.redtide.whoi.edu/pfiesteria/
This Woods Hole Oceanographic
Institute Web site contains information on molecular probes/HMA/FISH assay and a
summary report entitled "Molecular Approaches for Identification and
Environmental Detection of Pfiesteria piscicida and Pfiesteria-like
Dinoflagellates" (meeting held at the Columbus Center; University of Maryland,
Baltimore, MD, September 1999).
http://ccmp.bigelow.org/
This Provasoli-Guillard National
Center for Culture of Marine Phytoplankton (CCMP) Web site contains information
on dinoflagellate cultures characterized by several methods, including HMA, 18S
sequencing and PCR.
http://www.pfiesteria.org
This North Carolina State
University Applied Aquatic Ecology Laboratory Web site contains information and pictures of Pfiesteria sp.
Progress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.