Final Report: The Development of a DNA Based Specific Assay for Pfiesteria piscicida in Water and Sediments

EPA Grant Number: R827084
Title: 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: Hiscock, Michael
Project Period: October 8, 1998 through October 7, 2001 (Extended to August 31, 2002)
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 , Ecosystems

Objective:

The association of Pfiesteria-like dinoflagellates with fishkill 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. Through collaborative efforts during the entire project period, we utilized a methodology that determined full-length 18S sequence data for several Pfiesteria spp. and developed real-time polymerase chain reaction (PCR) assays for the detection of these species in culture, environmental samples, and sediment (surface and cores). These efforts have contributed to a better understanding of the role of Pfiesteria spp. in ichthyotoxicity (by implementing these assays in fishkill bioassays and environmental fishkills) and increased knowledge of their known distribution (through deployment in state monitoring programs that include water and sediment sampling and by testing samples collected in foreign waters by collaborating laboratories abroad).

Summary/Accomplishments (Outputs/Outcomes):

Sequence Derivation, Assay Development and Utilization in Determining Culture Clonality. During this project, the Oldach and Rublee laboratories have been instrumental in developing, validating, and implementing real-time PCR assays for detecting Pfiesteria spp. and other organisms in culture, environmental water, and sediment samples. To initiate this work, a matrix of known dinoflagellate 18S sequences (downloaded from GenBank) was designed. Since then, we have deposited several 16S and 18S sequences derived from characterized cultures, thereby adding to the database of known sequence data available to the research community. Full-length 18S sequences for Pfiesteria piscicida and Pfiesteria shumwayae were deposited in GenBank (AF077055 and AF218805, respectively). In addition, 18S sequence data for several other Pfiesteria isolates were generated and deposited in GenBank (AY033487, AF330600, AF330601, AF330602, AF330603, AF330604, AF330605, AF330606, AF330607, AF330608, AF330609, AF330610, AF330611, AF330612, AF330613, AF330614, AF330615, AF330616, AF330617, AF330618, AF330619, AF330620), including a strain isolated from northern European waters (AY033488; Jakobsen, 2002). Sequence data (18S and 16S) also were generated and deposited into GenBank for Karlodinium micrum (previously Gymnodinium galatheanum; AF272045, AF272046, AF272047, AF272048, AF272049, AF272050, AF272051, and AF272052; Tengs 2001) and Karenia brevis (previously Gymnodinium breve; AF172718 and AF172714). These data, along with our experience with molecular techniques, have initiated our collaboration with other laboratories to submit additional ECOHAB proposals.

These sequence data also have led to the development and validation of several real-time PCR-based assays for the rapid and specific detection of various organisms (P. piscicida and P. shumwayae, K. micrum, K. brevis, and most recently several Raphidophyte species such as Chattonella verruculosa, Chattonella cf. verruculosa, and C. sp., which detects Chattonella marina, Chattonella antiqua, and Chattonella Subsalsa). Our work in molecular methods has permitted us to become reference laboratories for other researchers in the field for determining culture identification and clonality. Traditionally, morphological methods were employed to identify and characterize species; however, with the advent of molecular tools, more taxonomists are relying on sequence data to support their conclusions regarding species classification. These methods have aided several collaborators in confirming culture identifications.

Deployment of Real-Time PCR Assays in Estuarine Monitoring Programs. The Oldach laboratory has been involved with Maryland's Department of Natural Resources' (DNR) Pfiesteria monitoring program since 1998. This program involves determining the presence of Pfiesteria spp. in samples collected on a routine basis and rapid response samples collected in response to fish and/or human health events. To date, we have assayed close to 5,000 environmental water samples for P. piscicida and P. shumwayae. With more than 4 year's worth of data, we have found that certain waterways of the Chesapeake Bay system are more conducive to the presence of Pfiesteria spp. In addition, there is a clear temporal distribution, with these species most commonly detected in the water column in late summer through early fall. Ongoing analyses will address correlations of species detection and environmental parameters. In 2002, the Maryland DNR expanded this assessment to assaying selected samples for K. micrum and Chattonella spp. For the 2003 monitoring season, we will be developing and implementing several assays for additional harmful algal species.

Both the Oldach and Rublee laboratories have been involved with Delaware's Department of Natural Resources and Environmental Control (DNREC) monitoring program. To date, we have screened more than 700 samples for the presence of Pfiesteria spp., and a select subset for K. micrum and Chattonella spp. Observations in Delaware appear to correlate with those in Maryland; certain areas appear to support Pfiesteria spp. populations and temporal distribution is generally in late summer through early fall.

In addition to maintaining relationships with Maryland and Delaware state agencies, the Rublee laboratory established collaborations with several other agencies interested in monitoring for Pfiesteria species and correlating those data with ecological parameters: 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, Texas Department of Parks and Wildlife, and the Florida Department of Environmental Protection. Through this effort, distribution of Pfiesteria spp. has been extended to the southern tip of Texas.

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 subsequent 18S sequencing, two cultures were confirmed as novel strains of P. piscicida). In collaboration with researchers at Horn Point Laboratory, P. piscicida also was identified in surface water samples from the Finland archepelago in the Baltic Sea. Efforts are underway in the Oldach laboratory to derive full-length 18S sequence data from this isolate and to compare that sequence to other P. piscicida isolates.

International collaboration also has been established between the Rublee laboratory and researchers in 17 other countries, who have provided samples for analysis from estuarine locations. P. shumwayae has been positively identified in samples from New Zealand and Australia, and P. piscicida has been found in coastal waters of Latvia. During October 2001, Dr. Rublee visited Australia and New Zealand, presenting seminars and meeting with government and university researchers. They discussed strategies to identify distribution and risks associated with Pfiesteria, and helped to get the PCR methods up and running at the Cawthron Institute in New Zealand to aid in ongoing studies of harmful algae. International collaborations are continuing with these and other countries.

Correlation of PCR Data With Human Health and Environmental Parameters

During this project, a Centers for Disease Control and Prevention (CDC)-sponsored cohort was initiated involving approximately 50 watermen on Maryland's Eastern Shore working in the Tangier Sound region. This CDC-sponsored project was an effort to better understand the distribution of Pfiesteria spp. and link that data to any complaints of human and/or fish-health events. The Oldach laboratory screened less than 2,000 samples collected by the watermen (2001 and 2002) for the presence of Pfiesteria spp. and C. cf. verruculosa (a brevetoxin-producer). Matched samples also were collected and frozen at -80°C for toxin analysis studies (being performed by the Eldefrawi laboratory at the University of Maryland (UMD) School of Medicine) involving the mammalian N-methyl-D-aspartate (NMDA) receptor. Another set of matched samples were frozen at -80°C and await brevetoxin analysis by Dr. Andy Kane (MD Aquatic Pathology Laboratory).

Currently, the Maryland DNR and Delaware DNREC are working to correlate physical parameters and PCR results from screening less than 5,000 samples as part of their monitoring programs to determine temporal and spatial distribution of Pfiesteria spp. in the Chesapeake Bay and inland bays. These data will not only aid in determining environmental parameters that support populations of Pfiesteria spp., but also will serve as a model for future monitoring of other harmful algal species.

Utilization of Real-Time PCR Assays With Sediment Samples

We devised 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 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 laboratory) have been working on a sediment core project (funded by a Maryland Sea Grant) with The Johns Hopkins University, specifically, Dr. Grace Brush. We have begun analyzing dinoflagellate DNA from dated strata of sediment cores collected from Indian River (DE), Pocomoke (MD), and Chicamacomico (MD). Initial PCR screening found P. piscicida present in surface sediment (Indian River and Chicamacomico cores) and at 4 cm depth (Chicamacomico core). P. shumwayae was detected at 16 cm (Pocomoke core). Furthermore, to date we have generated several hundred dinoflagellate sequence tags from various increments in the cores utilizing our dinoflagellate-specific PCR. Pollen-grain analysis and dating of these cores is being performed by Dr. Brush, and will be based on a variety of sediment core parameters. These investigations may allow us to draw inferences regarding relationships between the distribution and abundance of Pfiesteria-species and other dinoflagellates and environmental transformations occurring in these waters in the past.

This method of DNA extraction from sediment samples also was utilized by our laboratories during the past 3 years to assess presence of P. piscicida and P. shumwayae in surface sediment samples collected throughout Chesapeake Bay tributaries in a combined effort by Maryland DNR and Maryland Geological Survey to identify possible "seed beds" for these species. In screening these samples, we have observed the conduciveness of certain tributaries to support presence of resting stages of Pfiesteria spp. (presumably cysts or other benthic resting stages). Many of the tributaries in which Pfiesteria spp. were detected in sediment correlated to areas where these species were previously present in the water column (as determined by PCR and presumptive counts) and/or there were fish-health events (based on fish-health data collected during intense annual monitoring of these systems from April to October).


Journal Articles on this Report : 17 Displayed | Download in RIS Format

Other project views: All 90 publications 23 publications in selected types All 17 journal articles
Type Citation Project Document Sources
Journal Article 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)
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  • Full-text: AEM
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  • Abstract: AEM
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  • Other: AEM
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  • Journal Article Grattan LM, Oldach D, Tracy JK, Greenberg DR. Neurobehavioral complaints of symptomatic persons exposed to Pfiesteria piscicida or morphologically related organisms. Maryland Medical Journal 1998;47(3):127-129. R827084 (Final)
  • Abstract from PubMed
  • Journal Article Grattan LM, Oldach D, Morris JG. Human health risks of exposure to Pfiesteria piscicida. BioScience 2001;51(10):853-858. R827084 (Final)
  • Abstract: BioOne
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  • Journal Article Haselow DT, Brown E, Tracy JK, Magnien R, Grattan LM, Morris Jr JG, Oldach DW. Gastrointestinal and respiratory tract symptoms following brief environmental exposure to aerosols during a Pfiesteria-related fish kill. Journal of Toxicology and Environmental Health, Part A 2001;63(8):553-564. R827084 (2001)
    R827084 (Final)
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  • Abstract: Informaworld
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  • Journal Article Jakobsen KS, Tengs T, Vatne A, Bowers HA, Oldach DW, Burkholder JM, Glasgow Jr HB, Rublee PA, Klaveness D. Discovery of the toxic dinoflagellate Pfiesteria in northern European waters. Proceedings of the Royal Society of London Series B: Biological Sciences 2002;269(1487):211-214. R827084 (2001)
    R827084 (Final)
  • Full-text from PubMed
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  • Journal Article Lewitus AJ, Hayes KC, Willis BM, Burkholder JM, Glasgow HB, Holland AF, Maier PP, Rublee PA, Magnien R. Low abundance of the dinoflagellates, Pfiesteria piscicida, P. shumwayae, and Cryptoperidiniopsis spp., in South Carolina tidal creeks and open estuaries. Estuaries 2002;25(4):586-597. R827084 (Final)
    R826944 (2000)
    R826944 (2001)
    R826944 (Final)
  • Abstract: SpringerLink-Abstract
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  • Journal Article Moe CL, Turf E, Oldach D, Bell P, Hutton S, Savitz D, Koltai D, Turf M, Ingsrisawang L, Hart R, Ball JD, Stutts M, McCarter R, Wilson L, Haselow D, Grattan L, Morris JG, Weber DJ. Cohort studies of health effects among people exposed to estuarine waters:North Carolina, Virginia, and Maryland. Environmental Health Perspectives 2001;109(Suppl 5):781-786. R827084 (2001)
    R827084 (Final)
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  • Full-text: EHP
  • Abstract: EHP
  • Other: EHP PDF
  • Journal Article 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)
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  • Journal Article Oldach D. "Real-time" polymerase chain reaction. Gastroenterology 1999;116(3):763-764. R827084 (Final)
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  • Journal Article Oldach D. Regarding Pfiesteria. Human Organization 1999;58(4):459-460. R827084 (Final)
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    Journal Article Rhodes LL, Burkholder JM, Glasgow HB, Rublee PA, Allen C, Adamson JE. Pfiesteria shumwayae (Pfiesteriaceae) in New Zealand. New Zealand Journal of Marine and Freshwater Research 2002;36(3):621-630. R827084 (Final)
  • Full-text: Informaworld PDF
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  • Abstract: Informaworld
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  • Journal Article 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)
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  • Full-text: VA Academy of Science
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  • Journal Article 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)
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  • Full-text: EHP
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  • Journal Article Silbergeld EK, Grattan L, Oldach D, Morris JG. Pfiesteria: harmful algal blooms as indicators of human:ecosystem interactions. Environmental Research 2000;82(2):97-105. R827084 (Final)
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  • Abstract: Science Direct
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  • Journal Article Steidinger K, Landsberg J, Richardson RW, Truby E, Blakesley B, Scott P, Tester P, Tengs T, Mason P, Morton S, Seaborn D, Litaker W, Reece K, Oldach D, Haas L, Vasta G. Classification and identification of Pfiesteria and Pfiesteria-like species. Environmental Health Perspectives 2001;109(Suppl 5):661-665. R827084 (2001)
    R827084 (Final)
    R826791 (Final)
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  • Full-text: ResearchGate - Abstract & Full Text - PDF
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  • Journal Article 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)
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  • Journal Article Tengs T, Bowers HA, Glasgow Jr HB, Burkholder JM, Oldach DW. Identical ribosomal DNA sequence data from Pfiesteria piscicida (Dinophyceae) isolates with different toxicity phenotypes. Environmental Research 2003;93(1):88-91. R827084 (Final)
  • Full-text: Science Direct
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  • Abstract: Science Direct
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  • Supplemental Keywords:

    estuary, fish health, human health, Atlantic coast, molecular biology., RFA, Scientific Discipline, Geographic Area, Waste, Water, 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 sequences

    Relevant Websites:

    http://www.whoi.edu/redtide/pfiesteria/ Exit
    http://ccmp.bigelow.org/ Exit
    http://www.pfiesteria.org/ Exit
    http://www.dnrec.state.de.us/dnrec2000/index.asp Exit

    Progress and Final Reports:

    Original Abstract
  • 1999
  • 2000 Progress Report
  • 2001 Progress Report