2001 Progress 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 , University of North Carolina at Greensboro
Current 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, 2000 through October 7, 2001
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 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 are factors that have hampered progress in these endeavors. Through collaborative efforts during the entire project period, methodology was utilized to determine 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 fish kill bioassays and environmental fish kills) and increased knowledge of their known distribution (through deployment in state monitoring programs that include water and sediment sampling; by testing samples collected in foreign waters by collaborating laboratories abroad).

Phase-I

During Phase-I of the proposed project the investigators developed a molecular approach utilizing dinoflagellate-specific 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, Rublee et al. 1999). Our laboratories then began collaborations with other laboratories to determine clonality of dinoflagellate cultures. Also, work has begun with state agencies to build a database of environmental parameters related to the presence or absence of Pfiesteria spp. as a result of comprehensive monitoring programs.

Phase II

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); (2) SSU sequence determination of Pfiesteria-like isolates to expand the dinoflagellate sequence matrix; (3) deployment of a real-time quantitative assay for the presence of P. piscicida and P. 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 spp. 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 for detection of Pfiesteria spp. in surface sediment and sediment cores (i.e., cysts and other benthic life stages). These investigations are being undertaken 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, real-time PCR-based assays were developed for the detection of P. piscicida and P. shumwayae, and rigorously tested them for specificity and sensitivity (Bowers et al. 2000). Each year, these molecular techniques have been employed 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. Furthermore, deployment of these assays in ongoing monitoring programs has expanded the known distribution of Pfiesteria species throughout the United States and abroad.

Analysis of Sediment Samples

Cores. During this period, a method was devised for extraction of dinoflagellate DNA from sediment. The DNA is PCR-ready and may be used with our real-time Pfiesteria-specific assays. This has allowed for the incorporation of sediment sampling as part of the 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, work has continued on a sediment core project (funded by Maryland Sea Grant) with The Johns Hopkins University. Analysis has begun of 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 more than 250 dinoflagellate sequence tags have been generated from various increments in the cores utilizing our dinoflagellate-specific PCR (Oldach et al. 2000). Pollen grain analysis and dating of these cores will be performed 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.

Surface Sediment. This method of DNA extraction from sediment samples also was utilized by our laboratories during the past year to assess the presence of P. piscicida and P. shumwayae in surface sediment samples collected throughout Chesapeake Bay tributaries in a combined effort by Maryland Department of Natural Resources and Maryland Geological Survey to identify possible "seed beds" for these species. Approximately 144 surface sediment samples were collected in November 2000 and April 2001 (12 stations on 12 tributaries). P. piscicida was detected in 10 percent of samples, while P. shumwayae was present in 8 percent. 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). A second panel of surface sediment samples (n equals 138) representing 12 tributaries was collected between November 2001 and January 2002. P. piscicida was detected in 38 percent of samples, and P. shumwayae was present in 7 percent.

Additionally, during this period the Rublee laboratory supplemented work in North Carolina, assaying both water and sediment samples for the distribution of Pfiesteria species along coastal areas. During 2001, P. piscicida was detected in 2 percent of samples, and P. shumwayae was present in 11 percent, using direct PCR. Currently, these samples are being re-analyzed using real-time PCR assays which are more sensitive.

Analysis of Estuarine Water Samples

During this project period, relationships were maintained with state agencies (the Maryland Department of Natural Resources; the Delaware Department of Natural Resources and Environmental Control (DENREC); the Suffolk County [NY] Department of Health; the New York Department of Environmental Conservation; the New Jersey Department of Environmental Protection; the Virginia Department of Environmental Quality; the South Carolina Departments of Health and Natural Resources; the Georgia Department of Natural Resources; the University of Texas and the Texas Department of Parks and Wildlife; and the 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 (approximently 5 percent positive) and P. shumwayae (approximently 3 percent positive) during this report period. Sediment samples have shown a higher incidence of positive assays than water samples. The results also have 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 MD Department of Natural Resources. Monitoring approaches and partial analyses have been presented in oral and poster form at several conferences. Similar analyses also are being performed for samples collected in Delaware's inland bays.

Maryland. During the 2001 monitoring program by the Maryland Department of Natural Resources, 1,096 samples were analyzed for the presence of P. piscicida and P. shumwayae (12 percent and less than 1 percent positive respectively). Routine samples were preserved with 1 percent acidic-Lugol's solution, while samples collected in response to a fish and/or human health event (rapid response) were sent unpreserved overnight to our laboratories. This year, to identify possible discrepancies between results for the two sampling conditions, (i.e., impact of grazers, shipping, Lugol's solution, etc.) a matched preserved and unpreserved sample was collected for each rapid response event. Results for 179 sets matched, while 23 sets had conflicting results (i.e., Lugols' sample was positive/live sample was negative or vice versa). Further PCR work is underway to help explain these discrepancies. Aliquots of live samples collected during rapid response events were frozen at -80 oC for toxin analysis studies (being performed by the Eldefrawi laboratory at the University School of Medicine) involving the mammalian N-methyl-D-aspartate (NMDA) receptor.

This was the first year for screening samples collected by a cohort of approximately 50 watermen on Maryland's Eastern Shore working in the Tangier Sound region. This Centers for Disease Control and Prevention-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. Approximately 426 Lugol's-preserved samples were analyzed for the presence of P. piscicida (less than 1 percent positive) and P. shumwayae (0 percent positive). Watermen also collected live samples that were frozen at -20 oC, until transported to the Oldach laboratory where they were frozen at -80 oC for toxin analysis studies (being performed by the Eldefrawi laboratory at the University School of Medicine) involving the mammalian NMDA receptor.

Additional estuarine samples (n equals 96) were collected by the Horn Point Laboratory (Cambridge, MD) from transects of the Pocomoke River between May and October 2001. Analysis of these samples revealed no presence of either Pfiesteria species.

Delaware. Our laboratories also employed species-specific PCR assays on routine estuarine samples collected by the Delaware Department of Natural Resources and Environmental Control (n equals 133). Approximately 6 percent of samples were found to be positive for P. piscicida, less than 1 percent to be positive for P. shumwayae, and 42 percent positive for Gymnodinium galathanum (Tengs et al. 2001). In the Rublee laboratory, 150 water samples were analyzed for P. piscicida (9 percent positive) and P. shumwayae (2 percent positive). Aliquots of live samples collected by DENREC and sent to the Oldach laboratory were frozen at -80 oC for toxin analysis studies (being performed by the Eldefrawi laboratory at the University School of Medicine) involving the mammalian NMDA receptor.

Analysis of Cultures and Fish Kill Bioassay Samples

During the total project period, collaboration with North Carolina State University (NCSU) has continued and involved screening of many 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. A total of 118 NCSU samples were analyzed by PCR in 2001, with 26 percent of samples positive for P. piscicida and 38 percent positive for P. shumwayae. Ongoing Pfiesteria species screening and 18S sequencing also is being employed in our collaboration with Old Dominion University (ODU) 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, greater than 80 isolates have been sequenced, and these data will be used to complement morphological data obtained through scanning electron microscopy. Additional samples (n equals 31) received from ODU were assayed in 2001 for P. piscicida, P. shumwayae, G. galatheanum (Tengs et al. 2001) and Cryptoperidiniopsis brodyii (Oldach et al. 2000).

The molecular approach has been used to develop real-time PCR assays for other harmful algal species, including Gymnodinium galatheanum (Tengs et al. 2001), Chattonella spp., and Gymnodinium breve. In the development of the G. breve assay, our collaborative relationship with Florida Marine Institute (FMI) has been strengthened. Screening of greater than 35 cultures of Pfiesteria-likes and G. breve received from FMI has been coupled with 18S sequencing in support of morphological characterizations.

In 2001, the Oldach laboratory began collaborations with Dr. Carmelo Tomas (University of North Carolina Wilmington) to characterize various Chattonella spp. Dr. Tomas' expertise in isolation, culturing, and morphological characterization of Raphidophyte species has been coupled with our molecular techniques to determine full-length 18S sequence data. To date, a real-time group-specific PCR assay has been developed for the detection of Chattonella sp., and species-specific assays for Chattonella cf. vericulosa and a novel sequence derived from a Delaware environmental water sample. Ongoing detemination of assay specificity has involved screening and sequencing of several Raphidophyte cultures deposited at Provasoli-Guillard National Center for Culture of Marine Phytoplankton (Boothbay Harbor, Maine).

Detection of Pfiesteria in International Waters

International collaboration has continued with the University of Oslo, Norway 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 P. piscicida. These results were recently published in the Proceedings of the Royal Society of London (Jakobsen et al. 2001).

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 compare that sequence to other P. piscicida isolates.

International collaboration also has been established with 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, Principal Investigator 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.

Future Activities:

Our laboratories will continue to assay environmental water and sediment samples for P. piscicida and P. shumwayae. In particular, intensive collaboration with Maryland DNR will continue in 2002. 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 (greater than 100 samples per month) as part of their comprehensive monitoring program. In addition, these organisms will be assayed from sediment being collected during spring 2002 from several more 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.

Our collaborations will continue 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 a 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.

Based on the molecular work and strong collaborations built since the beginning of this project period, two ECOHAB grants were submitted in January 2002. The first, submitted with Co-Principal Investigators Oldach, Burkholder and Glasgow (NCSU), is entitled "Bacterial Populations Associated with P. piscicida and P. shumwayae Toxicity." The primary goal of the proposed research is to identify bacterial associates that are etiologically linked to the toxicity phenotype of P. piscicida and P. shumwayae. The hypothesis that bacterial associates (intra- or epicellular symbionts) share a direct or indirect role in toxicity will be tested using a broad array of molecular and dinoflagellate culture methodologies, including state-of-the art fluorescence activated cell sorting, Pfiesteria toxin detection assays, and standardized fish kill bioassays. Selective antibiotic treatments, bacterial transfer experiments, standard and halophilic bacterial culture and isolation methods, and broad-scale eubacterial and archael 16S ribosomal gene sequencing of cultures, with well-defined toxicity phenotypes, will be utilized. These investigations will enhance ongoing efforts to elucidate mechanisms of Pfiesteria spp. toxicity.

The second proposal, entitled "Toxin Production, Detection and Phylogeny of Chattonella species and Fibrocapsa japonica," was submitted with Co-Principal Investigators Oldach and Tomas. The primary objective of this research is to characterize Raphidophyte species by linking morphological identifications (SEM on cultured isolates, both known and novel) with toxicity data (highly sensitive ELISA for detection of brevetoxins) and molecular data (full length 18S sequences). The study, if funded, would confirm the identity of toxin(s) produced, provide verified molecular tools for the early and accurate detection of these species in local waters, and aid in defining their bloom dynamics and assessing the potential risks related to the toxins they produce.


Journal Articles on this Report : 8 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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  • 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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  • 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)
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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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  • 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 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)
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    R825551 (Final)
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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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    R825551 (Final)
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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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  • 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 sequences

    Relevant Websites:

    http://www.redtide.whoi.edu/pfiesteria/ Exit
    http://ccmp.bigelow.org/ Exit
    http://www.pfiesteria.org Exit
    http://www.nsgo.seagrant.org/research/hab/sg_hab_plan.html Exit

    Progress and Final Reports:

    Original Abstract
  • 1999
  • 2000 Progress Report
  • Final Report