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2001 Pfiesteria: review of the science and identification of research gaps. Report for the National Center for Environmental Health, Centers for Disease Control and Prevention Jonathan Samett

Gary S. Bignami

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Citation/Publisher Attribution Samet, J., Bignami, G. S., Feldman, R., Hawkins, W., Neff, J., & Smayda, T. (2001). Pfiesteria: review of the science and identification of research gaps. Report for the National Center for Environmental Health, Centers for Disease Control and Prevention. Environ. Health Perspect, 109(suppl 5), 639-659. Retrieved from https://ehp.niehs.nih.gov/doi/10.1289/ehp.01109s5639 Available at: https://ehp.niehs.nih.gov/doi/10.1289/ehp.01109s5639

This Article is brought to you for free and open access by the Graduate School of Oceanography at DigitalCommons@URI. It has been accepted for inclusion in Graduate School of Oceanography Faculty Publications by an authorized administrator of DigitalCommons@URI. For more information, please contact [email protected]. Authors Jonathan Samett, Gary S. Bignami, Robert Feldman, William Hawkins, Jerry Neff, and Theodore Smayda

This article is available at DigitalCommons@URI: https://digitalcommons.uri.edu/gsofacpubs/319 Pfiesteria: Review of the Science and Identification of Research Gaps. Report for the National Center for Environmental Health, Centers for Disease Control and Prevention

Jonathan Samet,1† Gary S. Bignami,2† Robert Feldman,3† William Hawkins,4† Jerry Neff,5† and Theodore Smayda6† 1Department of Epidemiology, Johns Hopkins University School of Public Health, Baltimore, Maryland, USA; 2The Queen’s Medical Center, Honolulu, Hawaii, USA; 3Boston University Medical School (retired), Boston, Massachusetts, USA; 4Department of Coastal Sciences, Institute of Marine Sciences, University of Southern Mississippi, Ocean Springs, Mississippi, USA; 5Battelle Ocean Sciences Laboratory, Duxbury, Massachusetts, USA; 6Graduate School of Oceanography, University of Rhode Island, Kingston, Rhode Island, USA

In connection with the CDC National Conference on Pfiesteria, a multidisciplinary panel evaluated It has multiple forms, transforming itself into Pfiesteria-related research. The panel set out what was known and what was not known about a toxin-producing dinoflagellate when it adverse effects of the organism on estuarine ecology, fish, and human health; assessed the somehow senses the presence of fish. Its toxin methods used in Pfiesteria research; and offered suggestions to address data gaps. The panel’s apparently destroys the integrity of the skin of expertise covered dinoflagellate ecology; fish pathology and toxicology; laboratory measurement of fish, allowing the organism to feed; after feed- toxins, epidemiology, and neurology. The panel evaluated peer-reviewed and non–peer-reviewed ing, the organism transforms into a dormant literature available through June 2000 in a systematic conceptual framework that moved from the form. (Throughout this report, we refer to source of exposure, through exposure research and dose, to human health effects. Substantial “Pfiesteria toxin,” recognizing that the organ- uncertainties remain throughout the conceptual framework the panel used to guide its evaluation. ism may well produce more than one toxin. Firm evidence demonstrates that Pfiesteria is toxic to fish, but the specific toxin has not been We use the plural “toxins” in describing the isolated or characterized. Laboratory and field evidence indicate that the organism has a complex basis for supposing that more than one toxin life cycle. The consequences of human exposure to Pfiesteria toxin and the magnitude of the human health problem remain obscure. The patchwork of approaches used in clinical evaluation and exists.) There is debate as to whether a surrogate measures of exposure to the toxin are major limitations of this work. To protect public pathognomonic lesion occurs in fish. Humans health, the panel suggests that priority be given research that will provide better insight into the seem to be at risk when their activities lead to effects of Pfiesteria on human health. Key gaps include the identity and mechanism of action of the contact with toxin-containing water at the toxin(s), the incomplete description of effects of exposure in invertebrates, fish, and humans, and time of fish kills. Whether the toxin reaches the nature and extent of exposures that place people at risk. Key words: dinoflagellate organism, target tissues by dermal contact or inhalation ecophysiology of Pfiesteria, epidemiology, fish kills, fish pathology and toxicology, Henle-Koch is unclear. A diffuse syndrome has been postulates, ichthyotoxins, literature review, neurological effects, state of the science. — Environ described in individuals likely to have been Health Perspect 109(suppl 5):639–659 (2001). exposed to toxin; its components include der- http://ehpnet1.niehs.nih.gov/docs/2001/suppl-5/639-659samet/abstract.html mal irritation, respiratory and gastrointestinal symptoms, systemic symptoms such as fatigue and malaise, and impaired neuropsychological functioning. The Centers for Disease Control Overview* understanding of each element in this and Prevention (CDC) has offered criteria for The Pfiesteria organism was first identified framework: the ecological factors that drive possible estuary-associated syndrome (PEAS) and named in the early 1990s and its ability concentrations of the organism and its state; for the purpose of surveillance (1–3). to kill fish was documented at the time. the stimuli that lead to toxin production and While the scientific evidence on Pfiesteria Research on the effects of exposure to its the nature of the toxin; the mechanism of is only now being accumulated, driven by toxin on humans is more recent, sparked the toxin’s effect on fish; the circumstances funding made available over the last several largely by widely publicized fish kills in the and routes of human exposure; and the years, there are sufficient credible human case late 1990s and reports of adversely affected mechanisms by which human health is persons present at the scenes of the kills. adversely affected by the toxin. Sufficient cer- Consequently, the peer-reviewed literature is tainty is needed on each of these points to This article is based on a presentation at the CDC National Conference on Pfiesteria: From Biology to still limited and substantial research is in evolve policies that will assure protection of Public Health held 18–20 October 2000 in Stone progress at institutions in the Atlantic coast public health and the fish populations. In Mountain, Georgia, USA. states of Maryland, North Carolina, and addition, we need enhanced understanding †These authors make up the Peer Review Panel on the state of of the science concerning Pfiesteria. Virginia, and elsewhere. Dramatic reports of of the still poorly characterized effects of Address correspondence to R. Black, Battelle/ fish kills and illness among persons exposed Pfiesteria on human health. CPHRE, 2971 Flowers Rd. South, Suite 233, Atlanta, to water at the time of the kills sparked public Other reviewers have commented on GA USA. Telephone: (770) 451-0882, ext. 14. Fax: (770) 451-6612. E-mail: [email protected] concern and reviews by a variety of agencies many elements of this framework; their The panel thanks C. Gerczak, Johns Hopkins in the late 1990s. The primary data sources, reviews make clear that there are gaps in sci- University School of Public Health; M.H. Ratner, secondary reviews, and expert panel reports entific understanding, with attendant uncer- Boston University School of Medicine; and B. Skarpness and R. Black, Battelle Centers for Public were evaluated by this panel. tainty for decision makers, for each of these Health Research and Evaluation in Atlanta for their In approaching its charge, the panel elements. With substantial research now assistance in preparing this report. In addition, we turned to a toxicologically based framework under way, the panel views the timing of this thank H.G. Marshall, Old Dominion University; S. Shumway, editor of the Journal of Marine Biology and for conducting its review, characterizing the review as an opportunity to examine the gaps Ecology; H.B. Glasgow and J.M. Burkholder, North state of the science, and identifying research and the extent to which new research findings Carolina State University; M. Swinker, Brody School of gaps (Figure 1). Prevention of adverse effects will set aside uncertainties. Medicine, East Carolina University; and K. Hudnell of of Pfiesteria will need to be based in an Pfiesteria has proved to be a challenging the U.S. Environmental Protection Agency, for provid- ing copies of manuscripts and allowing us to cite forth- problem. Researchers have referred to the coming work. *Section author: Jonathan Samet organism as “ephemeral” and “phantom-like.” Received 14 February 2001; accepted 11 July 2001.

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Ecological drivers and nonpathogenic bystander; and c) after review, the panel first attempted to characterize • Nutrient loading isolation from affected people and growth in the extent of the evidence available, enumer- • Predation/prey • Climate? culture, the organism can induce the disease ating to the extent possible the number of (4). Koch applied these criteria in describing independent observations, e.g., cases of the causal link of the tubercle bacillus with human disease reported and the numbers of Pfiesteria presence, form, and abundance human tuberculosis. samples studied in the laboratory setting. The Concentration (cells/mL) Recently, Fredricks and Relman (5) panel judged that some reports included over- described an extension of these criteria to lapping data and attempted in its review to sequence-based identification of microbial document the evidence available. Fish Toxin elaboration pathogens. They specifically addressed Beyond this detailed enumeration of the Human activity • Recreational sequence-based identification of microbial data available, the panel considered the • Occupational Fish effects Human exposure pathogens, now possible with modern assay Henle-Koch postulates as appropriate for • Dermal techniques. Marshall and colleagues (6) modi- linking Pfiesteria to either fish kills or human • Inhalation fied the Koch postulates and applied them to disease. As a minimum, there should be doc- findings of fish bioassays for the toxicity of umented presence of the organism at the time Toxin dose to Pfiesteria. In analogy to Koch’s first postulate, of fish kills or, in the instance of human dis- • Nervous system? • Skin? they proposed that Pfiesteria must be present ease, the presence of a fish kill when persons • Other? at a fish kill or disease event. For the second who become ill were in contact with water. postulate, they suggested that Pfiesteria must Effects on fish can be addressed in the labora- Adverse be isolated from a fish-killing sample and then tory, thus fulfilling the last postulate, but outcomes grown in clonal culture. For the third, addi- human effects cannot be investigated experi- tion of the clonal Pfiesteria to healthy fish cul- mentally. There is anecdotal evidence in the Figure 1. A framework for addressing Pfiesteria and tures results in death. Finally, Pfiesteria must form of experience of persons exposed in the public health. be isolated from the experimentally induced laboratory who have become ill. However, fish kill and recloned. Their report describes establishing causal linkages between exposure reports to suggest adverse effects of Pfiesteria experiments intended to meet these criteria. to Pfiesteria and human disease is complicated toxin on people. The panel viewed this clinical Criteria for causality in relation to human by the nonspecificity of the clinical picture as evidence as sufficiently compelling to motivate disease have evolved substantially since Koch’s presently characterized. a research agenda that would comprehensively writings over 100 years ago. In the mid-20th The evidence on ecological determinants address the many gaps in our understanding century, substantial epidemiologic research of the organism’s presence and concentration of Pfiesteria and human health. was initiated on the emerging epidemics of the is also observational. The panel’s approach for “chronic diseases”—cancer, coronary heart interpretation paralleled that used for the Principles for Interpreting Evidence disease and stroke, and chronic lung disease. human health data. In addressing its charge, the panel reviewed There was soon recognition that the Henle- This report follows the panel’s framework evidence associating Pfiesteria with fish kills, Koch postulates were not appropriate for offered in Figure 1. We begin with a review toxin elaboration, and adverse effects on interpreting the evidence on these multifactor- of the ecophysiology of Pfiesteria and then human health; it also considered ecological ial, complex diseases. A set of guidelines shift to its effect on marine organisms. The data on the presence of Pfiesteria and changes evolved (4,7,8) that was applied, for example, subsequent section covers approaches to mea- in nitrogen and phosphorus concentrations in interpreting the evidence on smoking and surement and detection of the organism and driven by man’s activities. The various lines lung cancer in the 1964 Surgeon General’s its toxin. After that, we address human health of data were derived from observation and Report (9). The guidelines consist of the fol- effects, including clinical and epidemiological also from the laboratory setting. No single lowing elements: consistency of association, reports. Finally, we offer conclusions and sug- framework can be applied across disciplines strength of association, specificity of associa- gest directions for further investigation. for evaluating such diverse evidence for its tion, temporal relationship of association, and strength in establishing linkages of hypothe- coherence of association. These elements are Ecophysiology of Pfiesteria sized sets of causes with observed outcomes, intended to be criteria for judgment and not a piscicida* e.g., Pfiesteria with fish kills, waste effluents rigid checklist. They call for consistency of P. piscicida Relative to Other into rivers with the presence of Pfiesteria, or findings with replication and coherence with Dinoflagellates and Harmful Algal Pfiesteria toxin with human health effects. other relevant observations, including under- Blooms To identify causes of human disease, standing of pathogenesis. The argument for criteria for determining the causal nature of causality is strengthened by the finding of a Pfiesteria piscicida Steidinger & Burkholder associations have been proposed, beginning stronger level of association, less likely to be represents a novel group of dinoflagellates first with Koch’s postulates, offered at the end of attributable to chance or bias, and the pres- discovered in the 1980s, and collectively the 19th century for judging if an infectious ence of a dose–response relationship. termed “ambush predators.” (Throughout this disease were caused by a particular microbe as Specificity is generally not relevant to multi- report, we use “Pfiesteria” to encompass described by Evans (4). Koch’s postulates, caused human diseases. “Pfiesteria piscicida Steidinger & Burkholder,” sometimes referred to as the Henle-Koch pos- The scientific evidence on Pfiesteria is still “Pfiesteria shumwayae Glasgow & Burkholder,” tulates to acknowledge the contribution of limited, with only a small number of research and other as yet unidentified species of the Henle, Koch’s professor, included a) an groups involved and insufficient time for genus. We use the standard shortened forms organism needs to be present in every case of replication and validation of key findings. “P. piscicida,” “P. shumwayae” whenever we the disease and “under circumstances which Thus, regardless of the criteria applied for can do so without causing confusion.) P. can account for the pathological changes and causal interpretation, the paucity of the data piscicida appears to be unique among this clinical course of the disease”; b) the organism limits the strength of inferences that can be is not found in other diseases as a fortuitous drawn. Consequently, in carrying out its *Section author: Theodore Smayda

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group and among dinoflagellates generally in sexual reproductive cells having a single set ingest other microscopic plant and animal its life cycle, nutritional behavior, and toxicity, of unpaired chromosomes) (14). prey at the same time. During this killing although much remains to be learned (10,11). Under laboratory conditions in the period, the zoospores reproduce both asexu- The ichthyotoxic blooms of P. piscicida were presence of live fish, its sediment-dwelling ally (mitotic division) and by producing first described in 1992 (10) and the organism amoeboid and resting stages transform gametes that fuse to produce toxic planozy- was classified taxonomically in 1996 (12). rapidly into free-swimming flagellate stages gotes (actively swimming offspring formed by Knowledge of the life cycle and feeding behav- in response to unknown chemical cues sexual reproduction, i.e., the union of two ior of the organism is based primarily on secreted or excreted by fish (10). The gametes) (Figure 2). The presence of live fish research conducted in the laboratory and on induced (excysted) flagellate stages swarm is required both for completion of the sexual less extensive field research. into the water column and become toxic cycle and for toxin induction. Current understanding of the salient during their continued exposure to the fish- Upon fish death or their retreat, the toxic features of the life cycle and of the behavior of derived (sometimes shellfish-derived) chemi- zoospores and planozygotes transform into P. piscicida has been described in several peer- cal stimulants. The toxic zoospores gather (mostly) nontoxic amoeboid stages that gather reviewed publications (6,10,11,13,14). The together, alter their random swimming pat- onto the floating fish carcasses on which they organism’s polymorphic life cycle (Figure 2) tern into directed movement, doubling their feed for extended periods, and follow the sink- consists of three distinct life-form stages—fla- swimming speed in the process (to 670 µm ing fish remains to the bottom sediments. Not gellated, amoeboid, and encysted—that live sec–1; median), and commence predatory all toxic zoospores and planozygotes transform in bottom sediments or as free-swimming behavior directed toward targeted fish. into amoebae. Some encyst and sink into bot- organisms in the water column. These stages The toxic zoospores produce a neurotoxin tom sediments; a lesser number revert to non- involve at least 24 size, shape and morpho- of unknown structure, soluble in water, and toxic zoospores that remain in the water typic variants, ranging from 5 to 450 µm in which may be liberated as an aerosol under column. The proposed 24 stages of the com- size. The stages include rhizopodial, filose some conditions. Fish are first narcotized by plex life cycle are based on laboratory observa- [i.e., the star amoebae in Figure 2; (15)] and the toxin, die suddenly, and slough off tissue, tion. Although several stage transformations lobose amoebae; toxic and nontoxic which the attacking zoospores consume by have been documented photographically, zoospores (asexual flagellated spore); cysts of sucking out the cell contents through the photographic or videotape documentation for various structure; and gametes (mature attached peduncle. The zoospores sometimes other proposed phases has not been published.

Planozygote (toxic) Water Toxic zoospore

Gametes

Rhizopodial amoeba

Toxic cyst

“Star” amoeba Amoeboid gametes Encysting (toxic?) amoeboid

Encysting amoeboid “Star” large amoeba (toxic?) NTZ NTZ

Small New cyst amoeba (toxic?)

Amoeba Cyst (toxic?)

Amoeba Large lobose Sediment Cyst Small amoeba (toxic?) (toxic?) Cysts amoeba (toxic?)

Cyst

Cyst Cyst (toxic?)

Figure 2. Schematic of the complex life cycle of P. piscicida with (+) and without (–) live finfish. Other environmental controls are indicated by: A, presence of flagellated algal prey; N, nutrient enrichment (e.g., organic or inorganic N and P); S, environmental stress such as shift in salinity, temperature, or physical disturbance. NTZ, nontoxic zoospore. Adapted from Burkholder and Glasgow (11) with permission from the American Society of Limnology and Oceanography.

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Neither is molecular confirmation available to advocated for Pfiesteria can be raised in rela- swarmers (19). The 24 stages presently recog- demonstrate that each of the proposed stages tion to the peer-reviewed and non–peer- nized in P. piscicida’s life cycle are consider- has the same genome. reviewed literature, including: ably less than the 38 stages recorded for Other dinoflagellate species have multiple • Some of the proposed “stages” in the H. ichthyophilum, the previously mentioned life stages that transition from bottom sedi- complex life cycle proposed for P. pisci- freshwater dinoflagellate that is also character- ments to the water column, feed on multiple cida are actually other organisms (“conta- ized by the nutritional versatility exhibited by trophic levels, are toxic to fish, “bloom” minants”) present in the P. piscicida P. piscicida (15). explosively under certain environmental con- cultures. Contamination of laboratory cultures of ditions, and are toxic to other marine organ- • Field events, toxicity features, and fish P. piscicida by other organisms cannot be isms and to humans. Pfiesteria’s distinctive kills attributed to P. piscicida have been ruled out and it is possible that some stages characteristics are its extraordinarily complex confused with the behavior of look-alike have been incorrectly identified. life cycle, nutritional diversity, trophic con- species, or are based on inadequate field Nonetheless, such complexity is neither trol of its life cycle and toxicity, and the way sampling. unique nor exclusive to P. piscicida among it kills fish prey. During fish kills attributed • P. piscicida outbreaks are consequences, dinoflagellates. Its complexity is not therefore to P. piscicida in natural habitats, and in con- rather than causes, of fish kills. an a priori reason to doubt the validity of the trast to blooms of many ichthyotoxic flagel- These challenges are addressed in the following life cycle as now described. Two additional lates, its lethal stages [i.e., toxic zoospores, sections. bits of evidence support this: During multi- planozygotes, large lobose amoebae (at tem- ple Pfiesteria-linked fish kills, several of the peratures <15°C)] usually represent only a The Life Cycle Issue life stages depicted in Figure 2 have been minor component of the phytoplankton The life cycle of P. piscicida (Figure 2) is recorded within the water column. The community present in the water column. based primarily on documented transforma- dominant amoeboid stages, which are partic- Blooms of P. piscicida are very ephemeral— tions of isolated cells and populations studied ularly abundant on sediments regardless of generally only hours in duration, of relatively in the laboratory and exposed to various com- fish availability, were ubiquitous during the modest cellular abundance (>250 cells mL–1), binations of temperature, salinity, nutrient fish kill events (20). More quantitatively, and rarely discolor the water mass (15). Of addition, prey availability, and live and dead analyses of ribosomal DNA sequences particular interest, P. piscicida is the first fish exposure (11,13,14). Life-cycle transfor- demonstrated that four dinoflagellate species dinoflagellate and the first harmful bloom mations appear to be highly sensitive to habi- (P. piscicida, a Pfiesteria look-alike species, a species observed to attack prey spanning all tat conditions, often occurring rapidly and cryptoperidiniopsoid sp., and the obligate trophic levels of estuarine food webs, from producing structurally similar stages despite fish parasite Amyloodinium ocellatum) are bacteria to fish (16). different life cycle pathways. Most dinoflagel- closely related members of an early ancestral The complexity of the life cycle and lates that spend part of their life cycle in the group (Blastodiniphyceae) having parasitic unusual behavior ascribed to P. piscicida raise water column and part in bottom sediments tendencies (21). Litaker et al. (22) also the following questions: Are the reported eco- are biphasic, with swimming and resting reported sequence data for P. piscicida, but physiology and trophic impact of this appar- stages. Pfiesteria is notable because it is appar- uncertainty surrounds this GenBank entry. ently novel dinoflagellate compromised by as ently triphasic, including flagellated, amoe- yet imprecise (inconclusive) methodology and boid (three distinct types), and resting Problem of Identification and investigation? Or is P. piscicida a particularly (encysted) stages and as many as 24 distinct Look-Alike Species problematic species, one that warrants contin- life-form variants. Two species currently form the Pfiesteria ued special research attention and public P. piscicida is not the first dinoflagellate complex: P. piscicida and a new species to be health concern? In asking these questions, the shown to produce amoebae that dominate its named P. shumwayae. A Pfiesteria sp. has been panel recognizes that the context of the evi- life cycle or the first to exhibit a triphasic life implicated in a fish kill in a marine home dence in which they are asked comes from cycle (17,18). Haidadinium ichthyophilum, a aquarium (23). This fish kill cannot be attrib- only a decade of research. freshwater dinoflagellate ectoparasitic on uted definitively to Pfiesteria because other Several scientific challenges to the validity three-spined stickleback, has four distinct parasitic dinoflagellates were identified in the of some key ecophysiological features amoeboid stages, cysts, and “gymnodinioid” tank and because the Pfiesteria-like organism disappeared before it could be examined and classified taxonomically. A combination of microscopic techniques and fish bioassay is required to detect P. piscicida in its various life stages (see below, “Measurement Tools for Detecting the Pfiesteria Organism”). Many small gymnodinioid dinoflagellate species look like P. piscicida zoospores under light microscopy. Taxonomic identification requires scanning electron microscopy (SEM) (12,18) (see below, “Scanning Electron Microscopy”). P. piscicida is easily overlooked in fish kills that it may have induced, for several rea- sons. Its toxic zoospore stages closely resem- ble other small, nontoxic species. Its small Figure 3. The dinoflagellate P. piscicida. (A) Apical view with epithecal plate tabulation and pattern. (B) Oblique flagellated forms tend to preserve poorly in ventral view showing four sulcal plates; the s.a. plate ties under the 1’ and 5” plates. (C) Dorsal view. Views B and C the fixatives routinely used in phytoplankton show the complete hypothecal plate tabulation and pattern. Scale bar = 7 µm. Reproduced by permission from analyses. When preserved, its amoeboid Journal of Phycology (12). stages often resemble organic debris, and its

642 VOLUME 109 | SUPPLEMENT 5 | October 2001 • Environmental Health Perspectives Pfiesteria: review of the science and research gaps

colorless heterotrophic cells blend into transformed into toxic zoospores when live predominate in moderately saline habitats “counting chamber” surroundings (15). fish were added (11). (5–18 ppt). Laboratory and field data suggest Proper identification requires SEM, an In summary, traditional methods of that 15 ppt is the optimum salinity for toxic- approach generally not used in field monitor- analysis typically cannot be applied to detect- ity of P. piscicida, with toxic outbreaks (as ing. The optimal period for detecting lethal ing and monitoring P. piscicida’s involvement zoospores) most frequent at ≥26°C within its Pfiesteria stages is during the fish kill (10), in fish kills for three reasons: a) its cryptic bloom temperature window, which ranges but sampling is usually done after the fish kill appearance, low abundance, and multiphasic from 10 to 33°C (15,20). Low temperature is reported and rarely conducted during the life cycle; b) its explosive, ephemeral bloom has also been shown to induce toxicity of active die-off period. events; and c) the existence of morphologically amoeboid stages. Addition of live fish or Cryptic occurrences prior to fish kills are similar but ecophysiologically distinctive shellfish (flounder and scallop) to experimen- another problem. Pfiesteria’s presence may be Pfiesteria-like species. These complications, tal aquaria held at 10–15°C triggered overlooked because of its low abundance or and discrepancies in fish bioassay results used ichthyotoxicity in the lobose amoebae that life cycle stage. A Neuse River, North to quantify P. piscicida involvement, have are the predominant life cycle stage of Carolina, experimental study was hampered fueled controversy. A rigorously standardized Pfiesteria in that temperature range (11). by chronic low abundance of the biflagellate fish bioassay process has only recently been Laboratory and field results indicate that zoospore stage (frequently <6 cells mL–1). used to replicate and confirm key findings con- P. piscicida is highly sensitive to water-column Abundance of other phytoplankton organ- cerning the ichthyotoxicity of P. piscicida (6). mixing and shear (11). In the laboratory, its isms of similar size ranged from 103 to 104 These gaps and limitations of the available flagellate stages encyst within minutes to cells mL–1 (24). Dead fish often are carried to evidence are a legitimate rationale for question- hours when exposed to rapid filtration or agi- regions outside of the active kill area by tidal ing the proposition that Pfiesteria transforms tation. Even gentle swirling of cultures can and other currents, blurring interpretations and then releases a fish-killing toxin. Published induce transformation. Field populations tend based on analyses of watermass properties in comments include rejection of the idea that to be evenly distributed within the water col- samples collected in the “dead zone.” P. piscicida blooms cause fish kills, and strenu- umn during calm weather unless tracking tar- The rapid stage transformations and ous denials that the organism may pose signifi- geted fish. During wind-induced mixing, population dynamics that characterize P. cant threats to natural fish stocks or human zoospore populations collect into lenses and piscicida contribute to these detection and health (24,27,28). Some of these objections cluster at or near the bottom. identification difficulties. Time constants of (27) are basically polemical arguments flawed Toxic Pfiesteria zoospores have no apparent life-cycle transformations are consistent with by scientific misinterpretations that have been optimal irradiance level and do not exhibit the ephemeral, rapid response, “sudden pointed out by Lewitus et al. (29) and Oldach diurnal activity in the laboratory. They actively death” syndrome described for ichthyotoxic (30). The more substantive points raised by kill fish at night or in full sunlight (11). Given Pfiesteria events. Upon stimulus, zoospores Pinckney et al., and Stow are addressed in their heterotrophic nature, this apparent indif- can transform within minutes into filose subsequent sections of this report. ference to irradiance is not surprising. amoeboid stages (Figure 2) (18). Flagellated Although the life history stages of P. pisci- stages subjected to shear can encyst within Distribution and Ecophysiology cida are controlled primarily by the availabil- minutes to hours (11). Nontoxic zoospores of P. piscicida ity of fresh fish secretions/excretions or fish become lethal within 3–4 hr upon exposure to P. piscicida is distributed in estuarine waters tissue, the availability and utilization of alter- live fish (15), and they are capable of growth throughout the mid-Atlantic and southeast- nate prey and dissolved nutrient energy rates that double the population in 15–21 hr ern United States, from Delaware Bay to sources are also important nutritionally (25,26). Recently formed cysts (hours to days Mobile Bay, Alabama, and may be found as (11,13,29,34). The nutritional versatility of in age) can excyst and yield toxic zoospores far north as Long Island, New York (31). It is P. piscicida is remarkable. Zoospores consume within 15–20 min after exposure to live fish best known for its association with fish kills bacteria, microalgae, microfaunal ciliates, ery- (11). In culture, toxic zoospores of P. piscicida in the Albemarle–Pamlico Estuarine System throcytes, and fish tissue; amoebae are equally and two tested Pfiesteria-like species killed (Pamlico and Neuse Rivers, North Carolina) versatile. When deprived of fish tissue, healthy fish within minutes after exposure (13,15,32), where it was implicated as the zoospores and amoebae (in culture) can sur- (15). Toxic zoospores sharply declined in causative organism in 50% of 35 major fish vive on a diet of microalgal prey but then abundance within 1–2 hr after fish death as a kills (103–109 fish) observed during exhibit prey selectivity and differing growth result of their transformation into cysts or 1991–1993 (15). Within the Chesapeake rates based on the microalgal species ingested amoeboid cells (10). Bay, it has been recorded from the Choptank [see Table 1 in Burkholder and Glasgow P. piscicida is also adapted to withstand and Patuxent River estauries in Maryland and (15)]. Zoospores consume microalgae, either prolonged periods of unsuitable growth con- the York River Estuary in Virginia; and it by phagotrophy or by attachment of their ditions, a feature that further compounds the and/or a related species is found in 7 of 10 peduncles to prey, followed by sucking out problems of its detection and association with eutrophic Florida embayments where fish cellular contents in saprophytic manner. putative fish kills. The lag period for excyst- kills have occurred (15,33). Within this dis- P. piscicida also can function as a ment upon stimulation by live fish varies from tribution, P. piscicida behaves as a warm- phototroph, carrying out photosynthesis by minutes to days to months, and increases with temperate estuarine species that occurs across utilizing the chloroplasts captured from cyst age. Cysts dormant for two years required a wide range of temperature and salinity but microalgal prey and inorganic and organic 6–8 weeks to produce toxic zoospores; less displays a distinct preference for relatively nitrogen and phosphorus. Laboratory obser- than 2 weeks if dormant for 2–8 weeks, and shallow, turbid, slowly flushed, nutrient- vations indicate that this expropriation and less than 30 min if only hours or several days enriched, moderately saline habitats (5–18 retention of the captured chloroplasts old (11). Lobose amoebae held in culture one ppt). An exception to this may be its recent (termed kleptoplastidy) can last for at least 9 year produced nontoxic zoospores when sup- discovery (via fish bioassay) in the pristine days (29,34). plied with a microalgal prey, and a Pamlico North Inlet Estuary of South Carolina (26). The nutritional versatility of P. piscicida is Sound, North Carolina, water sample rich in Pfiesteria-linked fish kills occur over a consistent with the polymorphism and differ- amoebae and kept in darkness for 4 months broad range of salinity (0–35 ppt) but ing habitat requirements of its life cycle

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stages. It is a “prey generalist” that can behave Pinckney et al. (24) concluded from into fish aquaria, and fish died. P. piscicida as an ichthyoparasite and survives periods of mesocosm experiments that Pfiesteria-like was identified in these cultures by SEM. fish deprivation by means of a complex life organisms in the Neuse River do not respond It seems reasonable to conclude that cycle and nutritional versatility. to inorganic nitrogen and phosphorus nutrifi- P. piscicida is highly tolerant of nutrient- Research has focused on the toxic cation, nor is their growth negatively influ- enriched waters, which appear to be a pre- “ambush predator” features of P. piscicida; enced by water column mixing. The authors’ ferred habitat, but neither laboratory nor field there is very limited information on the interpretation of their findings directly con- research has identified specific conditions extent to which it is itself predated. Mallin et tradicts laboratory experience with these eco- within eutrophicated habitats that are respon- al. (35) have shown that a rotifer and the physiological factors (11,16), which Pinckney sible for ichthyotoxic blooms. To conclude common estuarine copepod Acartia tonsa et al. dismiss in extrapolating their results to that eutrophication, per se, will lead to geo- readily grazed upon P. piscicida in short-term P. piscicida. Their study is compromised by graphical spreading and increased magnitude experiments. There is no information on the serious experimental flaws (control popula- of its bloom events would be premature. long-term effects of Pfiesteria blooms on these tions usually died off) and taxonomic uncer- or other grazers. Bay scallops and oysters did tainties, inadequate consideration of Pfiesteria Fish Kills: Cause or Consequence not strongly stimulate toxic activity of P. life cycle stages and their trophic regulation, of P. piscicida Blooms? piscicida, and scallops remained viable for 9- and a restricted data focus. That P. piscicida can become ichthyotoxic to 14-day test periods while filtering low con- In laboratory culture, P. piscicida responds upon exposure to live fish under appropriate centrations of toxic zoospores (~60 cells both via increased abundance and life-cycle laboratory conditions appears to be clearly mL–1) (20). At higher toxic zoospore levels transformation to enrichment with inorganic established. The reported threshold density of and in the presence of dying fish, scallops and organic nutrients (10,11,14,26). Under toxic zoospores required to kill fish is died. These data suggest that filter feeding some laboratory conditions, phosphorus- 250–300 mL–1. During 26 estuarine/coastal shellfish may help control the abundance of containing substances released by fish or fish kills linked to P. piscicida (20), observed P. piscicida, particularly when population direct phosphorus enrichment stimulate zoospore population densities ranged from densities are low. growth of zoospores. 270 to 35,360 cells mL–1 during fish kills. Given the complex life cycle of P. Zoospores that have “stolen” chloroplasts Zoospore densities in another fish kill were piscicida, the problems of detecting its pres- from microalgal prey take up inorganic and below the reported threshold. The various ence, its impacts on fish, and the limited avail- organic nitrogen at rates equaling ingestion life-cycle stages of P. piscicida were also pre- ability of quantitative field studies, little can of particulate nitrogen (prey N) by phago- sent during fish kills (11,32). be said about the ecological drivers of its pop- trophic stages. In natural settings, nutrient Some authors have argued that P. pisci- ulation dynamics, ichthyotoxicity, or spread- enrichment often stimulates the growth of cida is not the cause of, but a consequence ing potential. Attention has been focused on microalgae and other microbial loop com- of, these die offs (28,37). Stow’s reservations the hypothesis that nutrient loading of waters ponents on which P. piscicida can feed. The (28) are based primarily on his misinterpreta- by man’s activities is one determinant of capacity of P. piscicida for both direct tion that field sample data on cell densities Pfiesteria’s presence and abundance. Lewitus uptake (phototrophy) and indirect uptake and presence of Pfiesteria were the sole basis et al. (29) warned that continued and/or via ingestion of stimulated prey (phagotro- for inferring its active role in the observed increased nutrient loadings of coastal and phy) suggests that the response of natural fish kills. His largely statistical argument estuarine habitats may lead to an increase in populations of Pfiesteria to nutrient enrich- ignores the laboratory-based experimental the magnitude and geographical range of ment is very complex. The specific pathway evidence that established the organism’s toxi- P. piscicida toxic events. About 75% of the of any stimulation depends upon the nutri- city in the first place (11), confirmatory fish toxic outbreaks of Pfiesteria-like dinoflagellates tional state and life cycle stage of P. bioassays, and the presence of toxic life stages have occurred in nutrient-enriched waters (20), piscicida. The specific pathway of nutrient during the fish kills [see Tables 1 and 2 in with Pfiesteria-linked fish kills often clustering stimulation may be through enhancement of Burkholder et al. (20)], and may be moot in in North Carolina estuaries near sites exposed microalgal prey abundance, or direct stimu- light of subsequent confirmatory fish bioas- to discharge from wastewater treatment facili- lation by nutrient uptake from ingested say results by others (7,34). ties, fish processing plants, domestic animal prey. Pfiesteria may require a specific chemi- Blazer et al. (37) challenged the assertion operations, and phosphorus mining (16). cal “water quality” factor in its habitats—the that Pfiesteria is responsible for the ulcerative Mean nontoxic zoospore abundance bottom sediments and water column—to skin lesions purported to be diagnostic of (1,240 cells mL–1) at four wastewater sites complete its life cycle, undergo stage trans- P. piscicida involvement in fish kills. Their was 6-fold higher than at control sites (14). formation or emerge from dormancy. This histological analyses suggested to them that In the Neuse–Pamlico estuaries, where total factor may also be influenced by degree and fungal pathogenicity can induce the same phosphorus averaged ≥200 µg L–1 and total type of nutrient loading. ulcerative lesions in juvenile menhaden. Kjeldahl nitrogen levels ≥580 µg L–1, The various field and laboratory results, While fish skin lesions may not be a useful Pfiesteria was abundant. paucity of quantitative data, and nutritional proxy for the presence of Pfiesteria or its tox- Nutrification of a small estuary by acci- versatility of P. piscicida complicate field and ins, Blazer et al. do not dispute the ichthy- dental discharge of swine waste effluent was laboratory efforts to establish a definitive otoxicity of Pfiesteria, or do they dispute the followed three weeks later by a fish kill relationship between blooms of the organism evidence that P. piscicida has caused major (>104), during which toxic flagellate and and degree of anthropogenic nutrient enrich- fish kills. This is also discussed in the section amoeboid stages of P. piscicida were present ment. The recent discovery (26) of P. pisci- “Effects of Pfiesteria on Marine Organisms.” (36). This fish kill cannot be definitively cida in the highly flushed, pristine, and The preponderance of evidence from attributed to nutrient induced stimulation of low-nutrient North Inlet Estuary of South laboratory and field investigations supports P. piscicida because concurrent disruption of Carolina (where no major fish kills have been the proposition that P. piscicida has caused habitat conditions and altered bloom reported) suggests that the effects of nutri- fish kills in estuaries of Chesapeake Bay and responses in the normal microbial and ents on Pfiesteria may be site specific. Water the southeastern coastal regions of the United microalgal communities also occurred. samples from North Inlet were introduced States. The evidence supporting the argument

644 VOLUME 109 | SUPPLEMENT 5 | October 2001 • Environmental Health Perspectives Pfiesteria: review of the science and research gaps that blooms of the organism are consequences, the putative growth/toxicity stimulant(s) often were present in the granulomas and rather than causes, of fish kills is considerably and the ichthyotoxin gram negative, rod-shaped bacteria were pre- less persuasive. The behavior reported for P. • Assessment of the role of environmental sent in the lesions. Pathological lesions in piscicida is consistent with the considerable signals in controlling loss, or gain, in other organs and tissues from wild fish-kill global experience with other ichthyotoxic toxin-producing capability, life-cycle specimens have not been reported. algal bloom species, their impacts, and estab- transformations, and functional pheno- Ulcerative skin lesions like those described lished procedures applied by harmful algal type occurrences, along with determina- above have been considered to be caused pri- bloom researchers. tion of the accompanying physiological marily by fungal infections and termed “epi- characteristics of the life-cycle stages and zootic ulcerative syndrome” or “ulcerative Conclusions population strains mycosis,” common lesions of fish worldwide Compelling evidence indicates that, in experi- Such ecophysiological studies will help to and attributed to the fungal pathogens mental enclosures, exudates, and/or excre- resolve current ecological enigmas, knowledge Aphanomyces invadans and Saprolegnia sp. tions from fish stimulate blooms of P. of which is essential to clarify the fisheries and (37,41,42). However, after the discovery of piscicida, which then attack the fish by pro- human health issues attributed to P. piscicida’s Pfiesteria and the association of the dinofla- ducing an exotoxin, although the toxin has bloom events. gellates with fish kills, fungal infections seen not been isolated or characterized. Field in menhaden and other fishes from mid- observations, together with the laboratory evi- Effects of Pfiesteria on Marine Atlantic estuaries were considered secondary, dence, indicate that P. piscicida has been Organisms* opportunistic infections occurring subsequent responsible for major fish kills in mid- In both laboratory bioassays and field situa- to epithelial damage caused by exposure to Atlantic estuaries. Laboratory experiments tions, the toxic stages of P. piscicida and Pfiesteria toxins (14,37,39). Blazer et al. (37) have revealed P. piscicida to have a very com- Pfiesteria-like dinoflagellates affect every have challenged this assertion after histologi- plex life cycle consisting of toxic and nontoxic species to which they have been exposed, at cally examining about 150 ulcerated men- stages, which exhibit great nutritional diver- least 33 species of finfish and four species of haden from sites in the Chesapeake Bay area sity. These traits suggest a multiple-niche estuarine invertebrates (10,11,20,38). In this where fish kills and ulcerated fish were attrib- requirement and dependency on several section, we focus our review on studies deal- uted to P. piscicida. Fungal hyphae were pre- trophic levels. Laboratory nutrient enrich- ing with the toxicologic and pathologic sent both in deeply penetrating ulcers and in ment experiments have yielded growth effects of Pfiesteria spp., particularly in finfish. raised lesions with or without epithelial ero- responses consistent with the results of field Pfiesteria is toxic to shellfish such as blue sion. The authors contended that the men- studies that suggest a preference for nutrient- crabs (Callinectes sapidus), bay scallops haden lesions from the Chesapeake area enriched habitats. (Argopecten irradians), and eastern oysters corresponded to similar lesions reported The limited availability of quantitative (Crassostrea virginica) as well (20), but worldwide and caused by the A. invadans. An ecophysiological observations from laboratory detailed reports on those species are not avail- Aphanomyces sp. was cultured from the men- and field studies, together with taxonomic able. Whether in aquaria or in the wild, haden lesions, but Koch’s postulates have yet complexities, has impeded general acceptance Pfiesteria principally elicits neurotoxic effects to be fulfilled for this fungus (37). Thus, the of the ichthyotoxic affects, distinctive life and skin lesions in exposed fish in patterns roles of Pfiesteria and various fungi in the cycle, and ecophysiology ascribed to P. pisci- that are considered to be characteristic of the pathogenesis of skin lesions in fish from fish cida. Some critical scientific initiatives that dinoflagellate toxins (10,11). kills along the mid-Atlantic coast remain would help to resolve such uncertainty uncertain. include the following: Effects of Pfiesteria Toxin on Fish • Establishment of research-quality cultures in the Wild Effects of Pfiesteria Toxin in Laboratory for wide distribution among the scientific The link between Pfiesteria and kills of wild Exposures community for life cycle, physiological, fishes was made when water from a site of fish Pfiesteria toxins, rather than contact with or and ecological study kills involving Atlantic menhaden (Brevoortia ingestion of the dinoflagellate cells, are • Development of more effective and tyrannus) was bioassayed with tilapia responsible for effects in fish and inverte- quicker methods to distinguish P. pisci- (Oreochromis aureus and O. mossambicus) held brates (20). Laboratory studies have shown cida in each of its life cycle stages from in aquaria (10). Subsequently, Pfiesteria has that toxin elaboration depends on the pres- look-alike species, and to quantify their been implicated in fish kills and epizootic dis- ence of live finfish or their secretions that abundance, distribution, seasonality, and ease in estuarine fishes along the Atlantic stimulate transformation of Pfiesteria cysts or toxicity coast (10,20,39). The presence of fish, usually other nontoxic life stages to toxin-producing • Laboratory and field investigations, using large schools of menhaden, stimulates stages. Large numbers of live fish are required appropriate controls, to clarify the role of Pfiesteria to excyst and transform into toxic to stimulate excystment and toxin produc- organic versus inorganic nutrients, includ- stages. The toxic stages of the dinoflagellate tion in Pfiesteria in the laboratory (14). ing those in sewage and in wastes from release toxins to the water and the toxins Burkholder and Glasgow (14) supplied agro-industrial sources, in stimulating cause disorientation, respiratory distress, and Pfiesteria cultures in 40-L aquaria with growth and toxicity of P. piscicida, and death in fish and invertebrates (11,39). Skin 15–20 live, 5- to 7-cm tilapia per day to the role of these and nonnutritional fac- lesions attributed to Pfiesteria may be acute or maintain densities of more than 2,000 tors, including prey and predatory chronic. Typically, gross chronic lesions are cells/mL of toxic Pfiesteria stages. Even in dynamics, in regulating its in situ growth, well circumscribed ulcers with necrotic cen- swarming schools of menhaden, such densi- population dynamics, and seasonal cycles. ters, or round raised, friable red nodules (40). ties rarely occur in the field. The lag period • Rigorous evaluation of the linkage Microscopically, the lesions are marked by a for excystment of Pfiesteria cysts increased between the stimulation of blooms of P. chronic inflammatory infiltrate, granulomas with the age of the cysts; cysts that had piscicida, induction of toxicity, and and exposed necrotic muscle. Fungal hyphae remained inactive for more than about 170 substances produced by targeted fish, days could not be activated in the laboratory including chemical characterization of *Section authors: J.M. Neff and W.E. Hawkins by exposure to live fish. Yet in the field, the

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organisms presumably remain encysted for Research progress is slowed by lack of and tissue and organ distribution. more than 170 days every winter. Evidently, purified toxin with which to conduct bioas- Pathophysiological studies should be additional stimuli or environmental condi- says and develop antibodies. Despite the accompanied by chemical analyses aimed tions that have not yet been elucidated must complex effects of the toxin, few clear pat- at characterizing the toxin and its binding be required to stimulate toxic Pfiesteria terns of toxicity emerge, those being general sites (below). blooms and maintain high densities of toxic neurological and skin effects and death. The stages in the field. actual cause of death, even in acute exposures, Measurement and Detection Some evidence indicates that Pfiesteria is not known and there is, as yet, no pathog- of Pfiesteria and Pfiesteria-like produces more than one toxin (see “Chemical nomonic lesion. Extreme caution is needed Organisms and Their Toxins* Properties of Pfiesteria Toxins”). when attributing particular fish kills, espe- As discussed above, similar effects have cially fish lesions, to Pfiesteria. Whatever the Measurement Tools for Detecting been observed in both the laboratory and the outcome of the ongoing controversy concern- the Pfiesteria Organism field (43). Bioassays of Pfiesteria with aquar- ing the role of the fungus in the pathogenesis Significant progress has been achieved toward ium fish are difficult to conduct. Because of skin lesions in wild fish, the mere presence developing methods for specific detection and purified toxins are not available, bioassays of lesions in fish—whether or not the lesions differentiation of Pfiesteria spp. and Pfiesteria- depend on culturing or acquiring the organ- are associated with a fish kill—is not a valid like organisms (PLOs). Nevertheless, these ism and transforming the cysts to toxic stages indicator of an ongoing or previous Pfiesteria methods remain labor intensive and restricted that release toxins. Furthermore, there are bloom. Until field techniques for identifying to laboratory facilities and are not yet avail- considerable human safety concerns as well as the Pfiesteria toxin are available, work should able for routine field application. Initial field the need to add fresh, live fish during the be continued to understand the role of determinations are based on relatively non- bioassay to ensure toxin production. Aphanomyces spp. and possibly other infec- specific monitoring of fish kills, particularly To date, histological effects of Pfiesteria in tious agents in skin ulcers in wild menhaden. those in which affected fish display behavioral fish have been characterized and documented Neurotoxic effects are considered to be abnormalities and ulcerative lesions. The only for the skin (39). Damage to “myelinated indicative of Pfiesteria toxin effects in fish as standard method for associating P. piscicida neural tissue” has been suggested but not sub- well as in humans. However, little information with fish kills involves analysis by light stantiated (33). Noga et al. (39) reported that is available on correlative pathology of the ner- microscopy, followed by fish bioassay and in striped bass exposed to Pfiesteria skin dam- vous system in affected fish. There is a need for SEM. Morphological analysis by SEM age began within 8 hr, resulting in a nearly rigorous, complete (full tissue), time- and dose- remains the definitive method for speciation complete denuding of the epithelium within associated pathological assessment accompany- of Pfiesteria, but it is limited to laboratories 48 hr. Bacteria colonized areas denuded of ing laboratory bioassays and, as far as possible, that have the requisite equipment and exper- epithelium. In severe cases, the erosion in kills of wild fishes. To resolve diagnostic bias, tise. Recently described methods employing extended to the basement membrane (39). consideration should be given to having specific gene probes have facilitated more The pathogenesis for skin lesions in laboratory Pfiesteria-related lesions in fish reviewed by an rapid detection and differentiation of P. pisci- fishes is assumed be the same for skin lesions independent team of fish pathologists and vet- cida, the closely related P. shumwayae in wild fishes. This was true particularly for erinary pathologists who have expertise in fish (“species B”), and other PLOs. However, the menhaden, in which the skin damage com- pathology. Additional studies are needed of the development of immunochemical assays, bined with systemic immunosuppression, as physiological effects (i.e., osmotic and ionic reg- which might be used for onsite analysis, has indicated by splenic lymphoid depletion, ulation, respiration, endocrinology, neurophys- been hampered by researchers not having resulted in secondary bacterial and fungal iology) of Pfiesteria exposure to better Pfiesteria-specific antibodies. Further efforts infections in the skin ulcers (20,39). Fungal understand the mechanisms of toxic action of will be needed to produce methods capable of infections in menhaden skin lesions examined the dinoflagellate toxins in fish. selectively detecting toxic Pfiesteria, since by Blazer et al. (37), however, appeared to be In summary, additional research should none of the available assays, except for the primary lesions and not secondary infections. be undertaken to do the following: fish bioassay, can differentiate toxic from Skin lesions often precede severe systemic • Characterize the role of Aphanomyces spp. nontoxic Pfiesteria strains. effects and the appearance of neurological signs in causing skin ulcers observed in wild of toxicity. With exposure to Pfiesteria, fish menhaden and the potential interaction Light Microscopy become lethargic, with episodic hyper- of this fungus with Pfiesteria toxin in Light microscopy is typically used for prelimi- excitability; they also experience respiratory dis- affecting fish nary identification and enumeration of tress, often gulping air at the surface of the • Conduct full tissue pathological evalua- Pfiesteria spp. in water samples. Distinguishing water (10,11,20,38,39). They exhibit a poor tions of fish from laboratory bioassays of Pfiesteria from other species may be difficult fright response, disorientation, and loss of bal- Pfiesteria and in the wild fish from because in these samples Pfiesteria spp. often ance (44). The fish lose the ability to osmoreg- Pfiesteria-associated fish kills comprise a minority of the phytoplankton ulate; in salt water, blood osmotic pressure - Develop a detailed standardized population (20). Although light microscopy increases as concentrations of sodium, chloride, protocol for necropsy, histopathologic lacks the resolution to provide a definitive and potassium in serum increase toward those examination, and diagnostic criteria for result (12), biologists with sufficient experi- in the ambient seawater (39). White blood cell specimens from laboratories conducting ence can identify and quantify organisms counts may be depressed by 20–40% in fish fish bioassays or analyzing fish collected whose morphology is consistent with Pfiesteria exposed to Pfiesteria toxins (45). Menhaden from fish kills. (46). Preserved cell samples are observed at and striped bass eggs do not hatch in the pres- - Compile findings of these examina- high magnification with bright field, phase ence of the toxic stages of Pfiesteria (20,45). tions in a centralized database shared contrast, and differential interference optics among laboratories. (12). Counts exceeding 300 zoospores/mL in Conclusions • Investigate the physiological and toxico- water samples taken from active fish kills are Relatively little is known about specific effects logical effects of Pfiesteria toxin exposure of Pfiesteria spp. on marine organisms. in fish, especially neurophysiologic effects *Section author: Gary S. Bignami

646 VOLUME 109 | SUPPLEMENT 5 | October 2001 • Environmental Health Perspectives Pfiesteria: review of the science and research gaps

considered presumptively positive for possible entire 18S small subunit (SSU) ribosomal results. Nevertheless, this assay appears gener- involvement of Pfiesteria in a fish-kill event DNA gene has been sequenced for the proto- ally robust and was successfully used to screen (10,20,47). Staining with fluorescent dyes may typical P. piscicida (GenBank accession 170 estuarine water samples, collected from increase specificity (48) but does not obviate #AF077055) and P. shumwayae (GenBank New York to northern Florida, for the pres- the need to use SEM for definitive identifica- accession #AF218805) strains (50). Partial ence of P. piscicida. Of the 170 samples, 35 tion of the organism (12). Light microscopy sequences are available for the 18S SSU gene were positive, including sites where there was cannot differentiate toxic from benign of a PLO [(21); GenBank accession no historical evidence of P. piscicida-related Pfiesteria but does provide a rapid screening #AF080098] and for P. piscicida mitogen-acti- fish kills. tool to select samples that should be tested for vated protein kinase mRNA (GenBank acces- toxicity by fish bioassay (47). sion #AF227275). Portions of the internal Fluorescent in Situ Hybridization transcribed spacer regions I and II, the 5´ end Fluorescent in situ hybridization techniques Electron Microscopy of the large subunit ribosomal gene, nontran- employ fluorescently tagged gene probes to Electron microscopy (EM) provides sufficient scribed spacer regions adjacent to the riboso- label target DNA (or RNA) in intact cells. resolution to classify the species, P. piscicida, mal genes, and the cyclin box gene have been FISH has been adapted by Rublee et al. (31) in a new family (Pfiesteriaceae) and genus of sequenced but have not been published. These to detect and enumerate P. piscicida in water dinoflagellate (12). SEM studies have revealed gene sequences offer the means to develop samples using selected 18S SSU rDNA probe a multiphasic life cycle with polymorphic highly specific analytical tools for environ- sequences reported by this group for P. pisci- unicellular flagellated, amoeboid, and cyst mental testing. To date, all reported assays are cida PCR. Unlike the fish bioassay, FISH stages. Transmission EM of the flagellated based on the Pfiesteria SSU gene sequences. does not require culture amplification prior to form (i.e., zoospore) shows typical dinoflagel- These include polymerase chain reaction analysis, and it may be better suited than late ultrastructure, including mitochondria (PCR) assays, a heteroduplex mobility assay/ PCR to the analysis of dinoflagellate popula- with tubular cristae, endoplasmic reticulum, single-strand conformational polymorphism tions in complex matrices, such as sediment lipid bodies, trichocysts, Golgi apparatus, (HMA/SSCP) assay, and a fluorescent in situ samples (51), where Taq DNA polymerase mesokaryotic nucleus with condensed chro- hybridization (FISH) assay. inhibitors can affect PCR results (31,46). mosomes, large food vacuoles, microtubular The FISH technique is conceptually basket associated with a peduncle, and a Polymerase Chain Reaction straightforward, involving sample concentra- multimembrane thecal complex with vesicles Polymerase chain reaction is a method for tion by centrifugation, fixation with and thin plates (12,49). exponential DNA amplification that has paraformaldehyde, and hybridization with SEM is the standard technique for become the default method for DNA and two 5´-fluorescein–labeled probes. The morphological and taxonomic classification of RNA analysis. PCR uses paired (forward and hybridization reaction takes place in a pro- Pfiesteria. Culture amplified zoospores are reverse) gene probe primers in combination grammable thermal controller, and, following processed for SEM using a membrane-strip- with a thermostable DNA polymerase that a washing step, the labeled cell samples are ping or suture-swelling technique that reveals are cycled through a repetitive process of captured on a black polycarbonate filter for a characteristic number and organization of DNA denaturation, probe annealing, and observation under a fluorescence microscope thecal plates [see Figure 1 in Steidinger et al. primer extension. Typically, PCR products equipped with filters that permit detection of (12)]. Recent examination of cultured P. pis- are separated by gel electrophoresis and green FITC fluorescence as well as red cicida cysts by SEM X-ray analysis indicates detected with DNA binding dyes. chlorophyll-derived autofluorescence. Due to that cystic scales contain silica (6), suggesting Rublee et al. (31) developed a PCR assay the potential for nonspecific binding, positive that Pfiesteria represents a primitive dinofla- for detecting P. piscicida in environmental control–confirmed P. piscicida and negative gellate species. SEM is used to confirm the water samples, based on P. piscicida-specific control dinoflagellates are also carried presence of Pfiesteria zoospores in positive fish forward primers and a generic eukaryotic- through the process. Multiple-labeled probes bioassays, as well as to reconfirm species iden- specific 18S SSU ribosomal DNA reverse are used to compensate for compromised tification following cloning by micromanipu- primer. Four species-specific forward primers accessibility of the probes to targeted lation or flow cytometry (7,47). SEM plays (65 For, 110 For, 286 For, 301 For) were sequences, which can result in a failure to effi- an essential role in the diagnosis of Pfiesteria evaluated. DNA sequences of the resultant ciently label target cells or in low fluorescent in water samples but suffers from a number PCR products showed 100% homology with signal strength (52). Detection of FISH- of potential drawbacks. These include the P. piscicida (GenBank sequence #AF077055). labeled cells can be adapted to flow cytometry possible overgrowth of competing algae dur- Environmental sample DNA analyzed by for quantitative analysis of target gene label- ing culture amplification (50), the inability to PCR was isolated from filter-concentrated cell ing in combination with additional parame- directly distinguish toxic from benign samples by maceration in a buffered detergent ters such as size distribution. Pfiesteria, the need for specialized equipment solution followed by chloroform extraction Rublee et al. (31) used FISH, in conjunc- and facilities, and the requirement for staff and isopropanol precipitation. tion with a PCR assay described above, to with expertise in sample preparation, data Rublee and co-authors (31) noted potential identify P. piscicida as far north as Long acquisition, and results interpretation. difficulties associated with assaying field Island, New York, thereby extending the samples, such as cross reactivity against dinoflagellate’s known range from New York Genetic Diagnostics unrelated organisms that share the target to Mobile Bay, Alabama. The FISH probe Pfiesteria gene sequencing efforts have focused sequence, the presence of high levels of non- also enabled researchers to discern P. piscicida primarily on the ribosomal gene complex. target DNA that can interfere with primer from look-alike species (for example, P. Extensive databases exist for these genes [e.g., hybridization, and decreased assay sensitivity shumwayae, Gyrodinium galatheanum, GenBank at the National Center for due to Taq DNA polymerase inhibitors Cryptoperidiniopsis nov. gen.) and to estimate Biotechnology Information, National Library and/or non specific adsorption of extracted its abundance in water samples from a major of Medicine (see http://www.ncbi.nlm.nih.gov/ DNA to particulate matter. These concerns fish kill and toxic Pfiesteria outbreak that genbank)] and the phylogenetic relationships were validated, as in some samples only one occurred in North Carolina waters in 1998 can be deduced from these data (46). The or two of the primer sets produced positive (53). Many of the sites where P. piscicida was

Environmental Health Perspectives • VOLUME 109 | SUPPLEMENT 5 | October 2001 647 Samet et al.

detected were not associated with active fish universal eukaryotic 5´ and 3´ SSU primers. adsorption to surfaces, metabolism, or other kills or a history of fish health problems, sug- A 1,800 basepair sequence has been deposited factors. Sterile aquarium water filtrate has been gesting that P. piscicida may usually exist in a with GenBank for P. piscicida (accession shown to be nonlethal to fish after 48 hr at benign state (26). #AF077055) and for P. shumwayae (species B; ambient room temperature (11,38), but water accession #AF218805). samples stored frozen retain toxic activity in Heteroduplex Mobility/Single-Strand Based on these sequences, Oldach et al. rats through at least one freeze–thaw cycle Conformational Polymorphism Assay (50) developed highly selective PCR primers (58,59). Lipophilic toxin fractions adsorbed to Oldach et al. (50) recently reported a hetero- for P. piscicida (Ppisc108F/Ppisc311R) and C-18 chromatography resin and eluted with duplex mobility assay (HMA) capable of P. shumwayae (SpecB-forward/SpecB- acetonitrile retained potent cytotoxicity (60). detecting and differentiating DNA “signa- reverse). PCR assays incorporating these tures” of Pfiesteria spp. and PLOs. The primers have been used to screen environ- Methods for Detecting Pfiesteria Toxin method is based upon PCR amplification of mental water samples and mixed population Fish bioassay. The batch-culture fish bioassay highly conserved regions within dinoflagellate cultures suspected of containing Pfiesteria is the standard method for detecting exotoxin 18S ribosomal DNA gene sequences. spp. The P. piscicida PCR assay has demon- activity associated with Pfiesteria (6,10, Sequences amplified from target organisms strated high specificity as evidenced by 28,30). The assay is used to test unpreserved are then combined with a “driver” DNA a) negative assay results for more than 400 environmental water (or sediment) samples sequence amplified from Gymnodinium san- estuarine water samples in which “generic taken from sites where fish kill events are in guineum to form DNA heteroduplexes that dinoflagellate” DNA had been detected by progress after it is determined by light are separated by gel electrophoresis. Since PCR; b) negative assay results for 33 charac- microscopy that presumptive Pfiesteria DNA heteroduplexes migrate more slowly terized non-Pfiesteria dinoflagellate cultures zoospore counts of ≥300/mL are present. The through the gel matrix than homoduplexes, and a series of 28 Pfiesteria-like dinoflagellate fish bioassay has been described as “a process distinct banding patterns are produced when cultures that did not share the identical HMA rather than a rigid procedural ‘recipe’ to allow the target sequence differs from the driver pattern; c) negative results for the most the flexibility needed for optimizing detection sequence. Homoduplex (single-band) pat- closely related dinoflagellate species available, of toxic Pfiesteria from samples collected terns arise when the driver and target DNA P. shumwayae and Cryptoperidiniopsis sp.; and across a range of environmental conditions” share 100% sequence homology or if marked d ) sequence identity among all P. piscicida (6). Nevertheless, the assay requires a biohaz- sequence divergence prevents hybridization. PCR product amplicons examined. The anal- ard Biosafety Level 3 laboratory to prevent In some examples, the technique has been ogous PCR assay for P. shumwayae, also sub- potential human exposure to Pfiesteria toxins. shown to resolve single nucleotide differences jected to extensive testing, has been shown to The assay typically is carried out with between DNA fragments, illustrating its util- cross-react with an as yet uncharacterized juvenile (3–7 cm) tilapia, Oreochromis spp., ity for guiding DNA sequence discovery. organism. Thus, the P. shumwayae PCR under conditions that favor proliferation of By modification of the hybridization primer pair is highly selective but does not toxic zoospores. For example, this would conditions, it is possible to concurrently per- have absolute specificity. include replicate tilapia cultures, each with form the HMA and an SSCP. The SSCP is 2–15 fish, kept in covered aquaria main- based upon the principle that the 3-dimen- Chemical Properties of Pfiesteria Toxin tained at 18–24°C, under a 12-hr light/dark sional conformation of a single-stranded The chemical properties of Pfiesteria toxin are cycle (30 µEin m–2 s–1) with 15 ppt Instant DNA molecule has a specific sequence-based not well understood, primarily because the Ocean salts (Aquarium Systems, Mentor, secondary structure in a non denaturing gel toxin has not been purified and characterized. OH, USA) and aeration (6). Bioassays are matrix (54). SSCP can often further resolve Informal suggestions have been made that at initiated by introduction of Pfiesteria cultures dinoflagellate strains not differentiated by least three toxins exist, including two lipid- or environmental samples (water or sedi- HMA. In combination, the HMA/SSCP soluble toxins and one water-soluble toxin ment). Control fish cultures without P. pisci- assays provide a high resolution means to dis- (55). A water-soluble toxin that appears to cida are maintained under otherwise identical tinguish among Pfiesteria species and strains. target the nervous system was tentatively conditions. Aquarium water samples are The HMA/SSCP has identified mixed assigned an estimated molecular mass of taken at frequent intervals to measure dis- dinoflagellate cultures previously thought to 400–500 atomic mass units (amu) (56). One solved oxygen and ammonia levels, and to represent clonal cultures. of the lipid-soluble toxins, estimated to have a determine Pfiesteria and other microbial The banding patterns produced by molecular mass of 390 amu (55,56), has been counts. A minimum live fish density must be HMA can be used as a guide for gene attributed dermonecrotic activity that may maintained for optimal stimulation of toxic sequence discovery. For example, the PCR contribute to the skin ulcer formation com- zoospores (11). Therefore, dead fish are amplicons yielding an HMA banding signa- monly observed in Pfiesteria-associated fish removed from cultures and replaced with live ture characteristic of multiple SEM-con- kills (38). The other lipophilic toxin has been fish to sustain growth of toxic zoospores and firmed P. piscicida cultures were cloned, termed “lipid-soluble lethal factor” (55). inhibit transformation to amoebae or non– sequenced, and found to be identical (50). Purification efforts may have been com- toxin-producing cysts. This sequence was used to design PCR plicated by attempts to isolate toxin from The time to death of fish exposed to toxic primers yielding a near full-length sequence preparations derived from nonclonal Pfiesteria varies, apparently depending on of the P. piscicida 18S SSU ribosomal DNA Pfiesteria cultures (57). Individual Pfiesteria whether toxin biosynthetic pathways have gene sequence. The remaining sequence was strains could produce a unique toxin or com- been induced in the dinoflagellate population appended by sequencing PCR products plement of toxins and metabolites. Thus, by (undefined) stimuli elaborated by fish. amplified using a generic dinoflagellate PCR over time the spectrum of toxins harvested However, population densities of ≥250–300 primer and a universal eukaryotic 3´ SSU might change in concert with the population toxic zoospores per milliliter can produce primer. For P. shumwayae, a similar strategy dynamics of the culture. Toxin instability lethal concentrations of Pfiesteria toxins (16). was employed, in which full-length 18S gene may also have impeded successful purifica- Burkholder has classified Pfiesteria toxin sequences were PCR amplified from a tion. It is not clear, however, whether this production capacity into three phenotypic HMA/SSCP-confirmed clonal culture using instability is due to chemical degradation, categories (6,47,53):

648 VOLUME 109 | SUPPLEMENT 5 | October 2001 • Environmental Health Perspectives Pfiesteria: review of the science and research gaps

• Actively toxic (Tox A) strains are produc- Pfiesteria toxins. While intoxicated fish may samples were extracted with methanol/water ing ichthyotoxic substances that have display characteristic signs (lethargy, erratic and partitioned against hexane, then methyl- caused stress, disease, or death in fish. swimming behavior, hemorrhage, lesions) ene chloride. The methylene chloride frac- Environmental samples of actively toxic that provide a degree of specificity, the assay tion was dried and dissolved in dimethyl Pfiesteria are “primed” for toxin produc- is time consuming (up to 21 days), requires sulfoxide (DMSO) for cytotoxicity testing tion and produce a positive fish bioassay Pfiesteria culture amplification, cannot be against Caco-2 human colon carcinoma and (i.e., cause disease or death of test fish) used in the field, and lacks specificity with neuro 2A mouse neuroblastoma cells. Cell within 21 days (usually in 4–9 days). respect to toxin identification. A rapid and counts and morphological changes were used • Nontoxic (Tox B) strains are potentially specific method that can detect toxic as cytotoxicity assay end points. A partially toxic—they can be induced to produce Pfiesteria and/or Pfiesteria toxins at fish kill purified toxin preparation adsorbed to C-18 toxins that cause fish stress, disease or sites is needed to expedite management of cellulose resin, eluted with acetonitrile, dried, death, but they are not actively producing public health interests. and resuspended in DMSO was used to toxins; Pfiesteria/PLO species collected as Modified fish bioassay. Ramsdell and establish assay parameters prior to testing fish part of surveys (i.e., not in response to co-workers described a miniaturized 24-well tissue extracts. This preparation was reported reports of fish disease or fish kills) may plate version of the fish bioassay that they to induce cell rounding, membrane blebbing, produce positive fish bioassays, but time used in validation studies for a reporter gene cytoplasmic graininess, and membrane lysis to death is 8–10 weeks. cytotoxicity assay (below), and to monitor at the extraordinarily dilute level of 1 × 10–16 • Noninducible (never toxic) strains are chromatography fractions during purification g extract/mL. This represents unprecedented incapable of producing ichthyotoxins in procedures (62). The assay is conducted with cytotoxicity for a partially purified marine the presence of live fish or their fresh one 7- to 10-day-old sheepshead minnow, toxin and the authors cautioned that the materials. Cypronodon variegates, added per well contain- result must be verified with pure toxin (when Actively toxic Pfiesteria strains will gradu- ing 1 mL 25 ppt Instant Ocean. Following available). Extracts of fish tissue from the kill ally lose the ability to produce toxin when addition of toxin samples, fish are observed site were mostly noncytotoxic except for cultured over a period of months, even when for up to 2 hr for signs of intoxication. Even some of the Atlantic menhaden samples. cultured in the presence of fish (53). though this assay has not been thoroughly However, the authors cautioned that these Whether associated microorganisms have a validated, it may be useful as a rapid screen- results were not conclusive because Pfiesteria role in toxin biosynthesis is not known. ing method to aid in bioactivity-guided toxin could not be rigorously established as However, intracellular bacteria have been purification of ichthyotoxins. the cause of the fish deaths. shown to exist in P. piscicida flagellated and Cellular assays. Cellular assays have been Reporter gene assay. Reporter gene assays amoebae stages (11,12). The phylogeny of used to test Pfiesteria culture water, fish tissue rely upon specific gene induction in respon- these bacteria has not been reported, but and toxin purification fractions for the pres- sive cell lines stably transfected with reporter their presence raises the possibility that they ence of toxin activity. These assays include gene constructs. A reporter gene assay based play a role in toxin production, as has been cytotoxicity (viability) assays and a reporter on the inducible expression of the immediate proposed for Alteromonas/Pseudomonas-like gene assay. early gene, c-fos, has been described by Fairey bacteria associated with the dinoflagellate Cytotoxicity assays. Ramsdell and et al. (62). The AP-1 transcription factor, Alexandrium tamarense (61). co-workers used a colorimetric cytotoxicity consisting of Fos and Jun protein dimer The complete process for associating toxic assay for detecting cell survival and prolifera- complexes, coordinates cellular responses to Pfiesteria spp. with a fish kill event involves tion based on the dye MTT [3-(4,5- growth and stress stimuli, including toxins the following (7,47): a) initial field determi- dimethylthiazol-2-yl)2,5-diphenyl tetrazolium (64). Previously, the same group demon- nation that an active fish kill event is in bromide], which undergoes reduction in the strated that various neuronal cell lines differ- progress that may be associated with a mitochondria of living cells to form a purple entially express c-fos in response to toxin Pfiesteria bloom; b) sample collection and formazan product (63). Culture water, toxin exposure, conferring a degree of selectivity to transportation to laboratory facility; c) enu- purification fractions, and a residual water the assay. For this assay, GH4C1 and neuro meration of PLOs by light microscopy and fraction were tested against a panel of eight 2A (N2AC) cells were stably transfected with determination that potentially toxic PLO cell lines, including three mammalian neu- a gene construct consisting of the c-fos regu- concentrations (≥300 zoospores mL–1) are ronal cell lines, three finfish cell lines, and latory region ligated to the coding region of present; d ) determination by fish bioassay two mammalian epithelial cell lines. Assays the reporter element, firefly luciferase. The that cultured PLOs produce ichthyotoxins; were carried out in 96-well plates and colori- transfected cell lines GH4C1-A1 and N2AC e) speciation of fish bioassay dinoflagellate metric end points were determined in a were evaluated for Pfiesteria toxin-induced population by SEM and molecular assays, microtiter plate reader. A rat pituitary epithe- expression of the reporter gene. confirming the presence of Pfiesteria; lial cell line, GH4C1, was shown to be the Reporter gene activity was induced in f ) cloning and retesting by fish bioassay, most sensitive to diethyl ether and residual GH4C1 cells exposed to toxic culture water SEM, and molecular assays. water purification fractions, and was therefore containing live Pfiesteria cells, whereas the Fish bioassays can also be used to detect selected for development of a reporter gene N2AC cells were unresponsive. In N2AC Pfiesteria toxin preparations (as opposed to assay (below). cells, the marine toxins brevetoxin (PbTx-1) toxin produced in situ during the bioassay). Another cytotoxicity assay was developed and ciguatoxin (CTX-3C) induced c-fos- Culture water filtrate (0.22 µm) from actively to detect toxicity in fish tissue in an effort to luciferase expression, while saxitoxin inhib- killing Pfiesteria cultures has been shown to ascertain whether consumers could be at risk ited PbTx-1 induced reporter gene activation. retain full toxicity and kill fish within 3 hr because of consumption of fishery products In contrast, GH4C1-A1 cells were selectively (11). Concentrated aqueous toxin extracts exposed to toxin during a fish kill (60). Live responsive to Pfiesteria toxin preparations, killed fish within 20 min in a modified fish fish representing several species were col- and unresponsive to PbTx-1, CTX-3C, STX, bioassay system (below) (62). lected from the site of an ongoing fish kill, and domoic acid. This selectivity is attributed The fish bioassay remains the standard although it is unclear that Pfiesteria was to a higher complement of voltage-activated method for detecting toxic Pfiesteria and confirmed to be the causative agent. Tissue sodium channels in the N2AC cell line.

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The GH4C1-A1 reporter gene assay apparent instability of the toxins is another DNA sequences each and report findings in showed a concentration-dependent induction barrier. The methods that have been devel- about 15 min. One can imagine that if PCR of c-fos–luciferase expression over a range of oped for toxin detection, especially the probes to “toxicity genes” can be developed 30–300 Pfiesteria cells/mL and was used to reporter gene assay, offer potential means to that the HANAA will permit on-site detec- screen environmental water samples for the break through this circular conundrum. tion of both the organism and its toxin- presence of toxin-producing Pfiesteria. This Development of a high performance liquid production phenotype. assay was also useful for bioactivity-guided chromatography (HPLC) method, based on In summary, to address research gaps, purification of Pfiesteria toxins, as water soluble correlation of reporter gene activity with elu- future research should focus on the following: fractions with reporter gene activity coeluted tion times, could serve both analytical and • Developing and preserving a standard with ichthytoxic activity. Finally, the assay was preparative functions. It seems logical to sug- panel of ichthyotoxic water samples shown to be useful for detecting Pfiesteria toxin gest that such efforts have already been under- and/or extracts derived from clonal activity in biological fluids, including tissue taken, and one wonders if the toxins and the Pfiesteria cultures culture medium and human serum. “phantom” organism are equally ephemeral. • Promoting collaboration among laborato- Despite its demonstrated utility, however, Without purified toxin, it still may be ries developing analytical and purification certain features of the GH4C1-A1-based assay possible to develop highly sensitive and selec- methods by distributing the standard are problematic. For example, at higher con- tive bioassays such as receptor-binding assays. ichthyotoxic water sample/extract panel centrations, Pfiesteria toxins inhibited c-fos- However, this assumes that a specific receptor • Standardizing the fish bioassay methodol- luciferase expression and resulted in a exists and, if so, that toxins competing for ogy to facilitate interlaboratory repro- bell-shaped dose–response curve. Thus, sam- receptor binding can be identified—perhaps ducibility ples must be serially diluted over a wide range requiring a substantial screening effort. With • Developing and/or refining chromato- to assure that responses are detected within the purified toxin available, it should be possible graphic methods for toxin analysis and relatively narrow linear response range. Also, to develop a variety of analytical methods, purification using 5-fold concentrated Instant Ocean salts were such as HPLC and capillary electrophoresis, a) conventional detection [e.g. diode array shown to nonspecifically induce reporter gene and possibly antibody-based toxin assays. The ultraviolet (UV) detection] and expression. This prevents testing of concen- availability of purified toxin would also enable b) bioassay methods (cytotoxicity, receptor- trated aqueous samples unless the salts can be structural determinations to be made using binding assay, rapid fish bioassay) to removed. The possibility that other agents will nuclear magnetic resonance (NMR), mass guide method optimization nonspecifically induce c-fos–luciferase expres- spectroscopy, and perhaps crystallography. • Applying mass spectroscopic detection sion in GH4C1-A1 cells cannot be ruled out. Structural identification could then be used to methods to chromatographic procedures to design haptens for antibody production. estimate molecular mass of putative toxins Conclusions A need clearly exists for a field assay for • Applying NMR methods to (purified) Two critical gaps remain in what is known Pfiesteria, its toxins, or both. Antibody-based toxin samples for structural determinations about detecting the dinoflagellate Pfiesteria assays can be adapted to rapid qualitative or • Producing antibodies and developing and its toxins. First, despite the development semiquantitative devices such as lateral flow immunochemical methods to detect the of species-selective PCR assays, gene sequences “dipsticks.” Furthermore, such antibodies can Pfiesteria organism and Pfiesteria toxin in targeted in current tests do not differentiate be used in laboratory-based methods such as the field toxic from benign Pfiesteria populations. the enzyme-linked immunosorbent assay or • Identifying genes associated with toxin Second, the chemical identity of Pfiesteria flow cytometry. To date, however, there are biosynthesis using genomic screening toxins remains unsolved, preventing develop- no reports of antibodies specific to either the methods and/or analysis of gene expression ment of toxin-specific analytical methods and Pfiesteria organism or its toxins. A toxin- • Adapting PCR and/or FISH methods to field assays and limiting the ability to under- specific immunoassay would be especially devices that can be used in the field to take systematic toxicological studies. useful, as antibodies to the organism would detect toxin-producing phenotypes of Efforts to identify genes involved in toxin be unlikely to differentiate toxin-producing Pfiesteria biosynthesis may be facilitated by the rapidly and benign Pfiesteria. evolving technologies emanating from the Finally, recent advances in the develop- Human Health Effects: Human Genome Project. For example, since ment of portable PCR devices make it realistic Epidemiologic and Clinical it is known that Pfiesteria toxin production is to expect that PCR assays will soon be carried Studies* inducible in the presence of fish, it may be out with hand-held, battery-powered instru- possible to compare gene expression in ments. The Hand-held Advanced Nucleic Epidemiologic Observations “Tox-A” versus “Tox-B” phenotypes by such Acid Analyzer (HANAA) developed by Dean Epidemiology comprises methods and methods as differential display reverse Hadley and co-workers at Lawrence principles used to investigate diseases and transcriptase–PCR, representational differ- Livermore National Laboratory (Livermore, their causes in human populations. In investi- ence analysis, or serial analysis of gene expres- CA, USA) measures 5 × 8 × 2 inches and gating new syndromes like that caused by sion (65). Sequences of inducible mRNA weighs about 2 pounds (66). This device is Pfiesteria, epidemiologists establish case defin- transcripts might then be targeted to develop currently undergoing a 6-month validation itions and develop hypotheses about expo- RT-PCR assays that are selective for toxin- study by scientists from the U.S. Food and sures that may have causal roles. The first producing Pfiesteria. Drug Administration, CDC, University of phases of epidemiological investigation are The development of toxin purification Maryland, Utah Department of Health, Los often descriptive, involving characterization methods and development of well-characterized Angeles Emergency Operations Bureau, and of the affected individuals and obtaining toxin-specific assays are interdependently the Southwest Foundation for Biomedical information about exposures they may have linked. Thus, without purified toxin, reliable Research. The device incorporates TaqMan received. Building on this initial information, and valid assays cannot readily be developed; fluorescent probes that allow near-real time conversely, without the assays to guide purifi- PCR assays to be carried out. The instrument *Section authors: Jonathan Samet and Robert cation, toxin cannot readily be purified. The can simultaneously test four samples for two Feldman

650 VOLUME 109 | SUPPLEMENT 5 | October 2001 • Environmental Health Perspectives Pfiesteria: review of the science and research gaps

epidemiologists next carry out hypothesis- also offered a description of the clinical exposure to solvents. Performance on these testing studies, typically using studies of features of the associated syndrome (Table 1). instruments may be influenced by many fac- either the cohort or case–control design. The For the purpose of surveillance, the CDC tors, including use of alcohol or medica- cohort study involves follow-up of exposed has offered a definition of PEAS (1,2). The tions, age, education, and presence of and nonexposed individuals and observation definition incorporates contact with estuarine cerebrovascular disease. If exposed and non- for the occurrence of the outcome of interest. water, as well as clinical features (Table 1). exposed persons are not comparable for such For example, cohort studies are now under These definitions are notable for including potential confounding factors, a study using way in Maryland, North Carolina, and exposure as a defining element because the a neuropsychological test battery may give Virginia, each involving persons with high clinical features of the syndrome are nonspe- biased results. potential for exposure to Pfiesteria and its cific and not pathognomonic. toxin because they work on the waterways. In In applying these definitions in the Review of Epidemiologic Evidence a case–control study, exposures of persons context of an epidemiologic study, potential Case reports. A number of case series have with the outcome of interest are compared methodologic problems are evident. been reported; these reports range from anec- with those of similar persons not having the Participants in a study may be unaware of dotal, popular accounts of affected scientists outcome. This design has not yet been whether a fish kill has occurred or if there are (67) to more formally collected case series applied specifically to Pfiesteria-related symp- typical lesions on the fish. Unless they are able (Table 2) (68). As would be anticipated, crite- toms and illness. The cross-sectional design to track fish kills and document the presence ria for concluding that the illnesses were (sometimes referred to as a “survey”) has been of the organism, researchers may have diffi- related to Pfiesteria were variable across the used to characterize persons with Pfiesteria- culty linking activities at different places and reports and some of the reports provide little related health problems. In this design, obser- times to exposure. Unless they are educated clinical information. A series of affected per- vations are made at one point in time and and aware, PEAS may be missed by healthcare sons was described in Environmental Health consequently, causal relationships may not be providers who may not query persons with Perspectives and the Maryland Medical Journal apparent. This design has been used in possible symptoms as to their exposure. in 1997 and 1998 (70,71,74–77). From describing the health of persons with expo- The nonspecificity of the clinical picture information provided, it is difficult to assess sure to Pfiesteria and making comparison to is also a potential methodologic limitation of the number of independent cases described the status of unexposed controls. observational studies of the consequences of across the multiple articles in the issue. The The epidemiologic method is particularly exposure to Pfiesteria. Although case reports reported cases ranged from dramatic accounts informative if both exposure(s) and out- document relatively dramatic and severe acute of substantial cognitive impairment in come(s) can be sharply specified and mea- illnesses among exposed persons, research exposed laboratory workers (16) and water- sured with little error. Nonspecificity and studies have focused on more subtle and pos- men (71,72) to symptom episodes in persons measurement error typically weaken epidemi- sibly long-term consequences of exposure. with relatively casual contact with water ological data, most often resulting in a bias These potential effects are typically evaluated where fish kills took place or may have taken toward negative findings. Both potential limi- using standardized neuropsychological test place (75). These case series have proved tations have already been evident in research batteries that address learning and memory. useful for describing the elements needed for on Pfiesteria. The exact toxin remains These test batteries are also used, for example, a working case definition and as a signal of unknown, as does the route of entry into the in assessing the health effects of occupational the need for more formal investigation. body—inhalation, percutaneous, or inges- tion. The potential for exposure can be Table 1. Conditions of exposure and symptoms present in possible estuary-associated syndrome. described as contact with water at the time of Present in Present in fish kills, but such contact is only a surrogate a for the actual exposure to toxin, which can- Symptoms following exposure 1997 definition 1999 definition not yet be measured or quantified. The out- Memory lossb XX b come is also nonspecific and has multiple Confusion XX Symptoms develop within 2 weeks after confirmed exposure X clinical dimensions, all with many other pos- Health provider cannot identify another cause for symptoms X sible causes. The assessment of neurocogni- Acute skin burning at site of water contact X X tive functioning, an outcome of concern, Headachesc X requires neurologic evaluation and the use of <2 weeks sophisticated test instruments. To date, a ≥2 weeks X variety of such tests has been used. Skin rash X X Eye irritation Public health researchers have attempted <2 weeks X to address these vexing methodologic prob- ≥2 weeks X lems by developing standardized approaches Upper respiratory irritation for characterizing exposure and outcome. <2 weeks X Participants at a 1997 workshop sponsored ≥2 weeks X by the CDC proposed that exposure to estu- Muscle cramps <2 weeks X arine water might be characterized by a) fish ≥2 weeks X with lesions consistent with P. piscicida or a Gastrointestinal symptoms (nausea, vomiting, diarrhea, abdominal cramps) X morphologically related organism; b) a fish <2 weeks kill involving fish with lesions consistent with ≥2 weeks X P. piscicida or a morphologically related aConditions establishing exposure: 1997: Exposure to estuarine water characterized by any of the following conditions: a) fish with organism; and c) a fish kill involving fish lesions consistent with P. piscicida or morphologically related organism (MRO) toxicity (20% of a sample of at least 50 fish of one without lesions of P. piscicida or related species having lesions); b) a fish kill involving fish with lesions consistent with P. piscicida or MRO toxicity; or c) a fish kill involving fish without lesions, if P. piscicida or MROs are present and there is no alternative reason for the fish kill (1). 1999: Development of organisms and with no alternative reason for symptoms within 2 weeks after exposure to estuarine water (2). bSymptom must be present. cFor both 1997 and 1999 definitions, the fish kill (1). The workshop participants three or more of the remainder of the symptoms must be present.

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Table 2. Symptoms and clinical conditions among persons with direct contact with waters of Pocomoke Estuary, complain of episodes of “foggy memory” and Chesapeake Bay, Maryland and controls, by estimated exposure.a irritability lasting about 12 hr and precipi- High exposure Moderate exposure Low exposure Controls tated by strenuous exercise. Findings of a Symptoms and clinical conditions (n = 11) (n =7) (n = 4) (n =8) fluorodeoxyglucose positron-emitting tomo- Neuropsychological symptomsb 9 (82%) 6 (86%) 2 (50%) 1 (13%) gram (PET) of patient B were normal Headache 9 (82%) 5 (71%) 2 (50%) 1 (13%)* (Tables 3–5). Skin lesions 8 (73%) 4 (57%) 1 (25%) 1 (13%)† Electrophysiological studies. Peripheral Skin burning on contact with water 5 (45%) 6 (86%) 1 (25%) 2 (25%)‡ nerve electrodiagnostic tests [electromyogram Diarrhea 5 (45%) 4 (57%) 2 (50%) 1 (13%) (EMG) and nerve conduction studies] were Nausea/vomiting 7 (64%) 4 (57%) 0 1 (13%) Abdominal cramps 4 (45%) 4 (57%) 1 (25%) 1 (13%) done in two subjects (patients A and B) Joint pain 5 (45%) 2 (29%) 1 (25%) 2 (25%) described by Glasgow et al. (16) Evoked Muscle/leg cramps 8 (73%) 2 (29%) 1 (25%) 2 (25%) potential studies were done in Patient B. Eye irritation 6 (55%) 2 (29%) 2 (50%) 4 (50%) Findings for Patient A were reported to be Sinusitis 5 (45%) 5 (71%) 3 (75%) 3 (32%) essentially normal with mild electromyo- Shortness of breath 2 (18%) 4 (57%) 1 (25%) 2 (25%) graphic changes and normal sensory and Pneumonia 2 (18%) 1 (14%) 0 0 motor conduction velocities consistent with aData from Grattan et al. (68). bConfusion, episodes of distortion, new or increasing forgetfulness, or difficulties concentrating. minimal EMG evidence of motor axonopa- *p < 0.01, Fisher’s exact test, two tail, comparing high-exposure group with controls; †p < 0.05, Fisher’s exact test, two-tail, comparing high-exposure group with controls. ‡Control watermen reported seeing sea nettles in association with sensation of burning skin; thy and no evidence for Guillain-Barré affected individuals denied sea nettle contact. If two controls with complaints of skin burning are presumed to have had contact with syndrome. Decreased ankle reflexes were also sea nettles and are excluded, p < 0.05, Fisher’s exact test, two tail. seen in this patient. The studies in Patient B showed no evidence of either peripheral or Epidemiologic studies. Several studies of and of two control populations—115 crabbers autonomic neuropathy. The visual and brain- more formal design than the case series have working in other areas and 125 nonfishing stem auditory evoked potential studies in been carried out. In materials reviewed by the residents of the communities of the crabbers. patient B were normal although neuro- panel, the level of information concerning psychological testing was reported to support study design is variable and key aspects of Clinical Assessment and Neurotoxicology a diagnosis of amnestic syndrome involving design are not included in all instances. Clinical manifestations. The clinical data verbal more than visual modalities (16). The Reviews of the specific studies follow: found in the various reports vary in their com- electroencephalogram (EEG) of patient B and GRATTAN ET AL., 1998. This study (68) pleteness, but they do provide a perspective on that of a Chesapeake Bay waterman examined included cross-sectional observations on 24 symptoms in apparently exposed persons by Bever et al. (76) were assessed as normal. people having direct contact with the waters (Tables 3–5). Many of the reports cite non- Neuropsychological studies. Most reports of the Pocomoke and other estuaries in the specific symptoms of dizziness, eye irritation, presented descriptions of Pfiesteria exposure- Chesapeake Bay. An additional eight unex- and headache (16,74–77). Gastrointestinal associated changes in emotional state such as posed watermen were recruited for compari- complaints of diarrhea and abdominal pain irritability and lability of mood; cognitive son. A structured questionnaire was used to were common, as were respiratory complaints impairments including confusion, poor con- obtain information on exposure and the par- of wheezing, coughing, and shortness of centration, disorientation, and memory diffi- ticipants were then placed into strata of high, breath. Dermatological symptoms were also culties; motor impairments including ataxia, medium and low exposure (Table 3). frequent. Complaints of cognitive deficits were dysmetria, and dysarthria; and sensory distur- Participants were queried about symptoms also described, including memory impairments bances such as decreased ability to smell and a standard neuropsychological test bat- that developed within hours after exposure. odors and paresthesia. tery was administered that was considered The cognitive deficits and memory impair- Shoemaker (74) reports findings of appropriate for exposure to Pfiesteria. ments have been reported to subside sponta- neurocognitive studies for four patients. The SAVITZ ET AL. 1998; SWINKER ET AL. neously in some cases but to be aggravated by specific tests used in the evaluation were not 2000. This study (69,70) compared North strenuous exercise in others. Other reports described. Findings for a 56-year-old woman Carolina workers with and without potential have suggested that treatment with cholestyra- were markedly abnormal immediately after for exposure to Pfiesteria. Using maps of mine may improve clinical prognosis and has- exposure to Pfiesteria but returned to normal locations where distressed or killed fish had ten recovery, but these observations lack within 1 month after treatment with been found in 1997, the investigators proper controls (74). cholestyramine (2 months after cessation of enrolled gill-net and crab-pot fishermen Neuroimaging studies. Radiological exposure to Pfiesteria). A second patient exam- working the Pamlico, Neuse, and Trent imaging was done in four individuals ined by the group, a 33-year-old man, had Rivers and tributaries in North Carolina. (16,74,76). The magnetic resonance image memory impairments that persisted 1 year These workers (n = 19) and an additional (MRI) of a waterman examined by Bever et after exposure; these memory problems were four state employees with potential for expo- al. (76) was reported as normal. A patient reportedly improved by treatment with sure were enrolled as the exposed group. The with persistent cognitive deficits reported by cholestyramine and multivitamins. Neuro- unexposed cohort was selected with match- Shoemaker (74) had a computerized axial cognitive findings in a third patient, a 32- ing for age, gender, occupation, and educa- tomogram (CT) that was normal and a MRI year-old man, were reported as abnormal 3 tional level but were from the Outer Banks that revealed mild inflammation of the mas- months after cessation of exposure. Two of North Carolina. All participants had a toid and a possible polyp in the sinus. weeks after beginning treatment with detailed medical evaluation and completed a Glasgow et al. (16) reported that their patient cholestyramine, minimal improvement in his neuropsychological test battery. B had minimal changes in the hippocampus performance on neurocognitive tests had GRIFFITH ET AL., 1999. Griffith et al. (73) that they considered to be of borderline sig- occurred, although the patient was reported provide findings of a cross-sectional study of nificance. Patient B had cognitive deficits on able to return to work after initiation of this 253 North Carolina crabbers who worked in neuropsychological testing that resolved after therapy. Memory impairments were also doc- waters known to be home to dinoflagellates, 2 months, although the patient continued to umented in a 44-year-old man seen by this

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Table 3. Clinical manifestations reported among persons with suspected exposure to P. piscicida. Suspected source of exposure, neurological and dermatological symptoms, and signs. ID # Refa Age (years) Sex Exposure history Neurological symptoms and signs Dermatological symptoms 1(74)23MWater skier × 1 hr Memory, ataxia, dysarthria 1.5 - to 2-cm pruritic lesions with follicular eruption 2(74)26MSwimmer upstream from water skier None reported 1.5- to 2.5-cm lesions 3(74)30F Swimmer in vicinity of water skier None reported None reported 4(74)56F Dept. of Environment worker Memory problems Burning sensation, rash and sorting fish desquamation of skin 5(74)41MSampling shell fish beds Memory problems 3 lesions with discrete macular desquamation of left hand 6(71)47MFisherman Memory problems None reported 7(71)33MWaterman (commercial fisherman) Memory problems None reported 8(71)32MCommercial diver Memory problems Skin lesions 9(71)44MFisheries worker Memory problems Skin lesions 10 (16) <40 NR Marine scientist Concentration and memory problems, Skin lesions paresthesia, ataxia, 11 (16) <40 NR Marine scientist Memory problems, irritability, emotional Skin lesions lability, disorientation, dysarthria, insomnia, paresthesias 12 (16) <40 NR Marine scientist Disorientation, concentration and memory None reported 13 (16)NRNR5 laboratory personnel and 2 nearby problems, office workers None reported None reported 14 (16) >40 M Waterman (commercial fisherman) Memory problems, dysmetria, decreased smell in right nostril and positive palmar mental reflex (left) and positive globellar reflex None reported 15 (75,76)383 M Boaters 2/3 disorientation, concentration and 2/3 skin lesions; 1/3 burning sensation (mean) memory problems, on contact with contaminated water 16 (75,76)384 M Working with samples of infected 6/ 7 disorientation, concentration and 5/7 skin lesions; 4/7 burning sensation (mean) 3 F water/fish (8–20 hr/week) memory problems, on contact with contaminated water 17 (75,76)398 M Watermen (commercial fishermen) 9/11 disorientation, concentration and 8/11 skin lesions; 5/11 burning (mean) 1 F memory problems, sensation on contact with contaminated water 18 (78)4593 M Watermen, researchers, recreational None reported None reported (mean) 19 F water exposures 19 (72,73,77)40 55 M Fishing, handling fish with lesions, 47/55 memory loss, confusion 31/55 rash, burning sensation on (mean) 11 F boating, swimming contact with contaminated water Abbreviations: F, female; M, male; NR, not reported. aReference numbers in parentheses correspond to reference numbers in text.

Table 4. Clinical manifestations reported among persons with suspected exposure to P. piscicida. Gastrointestinal, respiratory, and nonspecific symptoms. ID # Refa Age (years) Sex Gastrointestinal symptoms Respiratory symptoms Nonspecific symptoms 1(74)23MNone reported Shortness of breath Dizziness, headache, stiff neck 2(74)26MNone reported None reported None reported 3(74)30F Diarrhea, nausea, abdominal cramps None reported Headache 4(74)56F None reported Cough, wheezing, reduced FVC and FEV Eye irritation on pulmonary function testing 5(74)41MAbdominal pain, diarrhea None reported None reported 6(71)47MAbdominal pain Cough, wheezing, Headache, eye irritation 7(71)33MAbdominal pain, diarrhea Cough, pneumonia Weight loss, pneumonia 8(71)32MAbdominal pain, diarrhea None reported Headache 9(71)44MAbdominal pain None reported None reported 10 (16) <40 NR None reported None reported None reported 11 (16) <40 NR Abdominal pain, nausea, vomiting Shortness of breath, chest tightness Eye irritation, headache, 12 (16) <40 NR Abdominal pain, nausea Shortness of breath, pneumonia, asthmatic Eye irritation, headache, joint pain bronchitis 13 (16)NRNRNone reported None reported None reported 14 (76) >40 M None reported Shortness of breath, chest tightness None reported 15 (68)383 M 1/3 diarrhea; 1/3 abdominal pain 1/3 shortness of breath; 3/3 sinusitis 2/3 headache, 1/3 joint pain; 2/3 eye (mean) irritation 16 (68)384M4/7 diarrhea, abdominal pain, nausea 4/7 shortness of breath; 5/7 sinusitis; 5/7 headache, 2/7 joint pain; 2/7 eye (mean) 3 F 1/7 pneumonia irritation 17 (68)398 M 5/11 diarrhea; 4/11 abdominal pain; 2/11 shortness of breath; 5/11 sinusitis; 9/11 headache, 5/11 joint pain; 6/11 (mean) 1 F 7/11 nausea 2/11 pneumonia eye irritation 18 (78)4593 M None reported None reported None reported (mean) 19 F 19 (72,73,77)40 55 M 28/55 nausea, vomiting, diarrhea, 37/55 upper respiratory tract irritation 4/55 headache, muscle cramps, eye (mean) 11 F abdominal pain irritation Abbreviations: FEV, forced expiratory volume; FVC, forced ventilatory contraction. aReference numbers in parentheses correspond to reference numbers in text.

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Table 5. Clinical manifestations reported among persons with suspected exposure to P. piscicida. Findings of neuroimaging, electrophysiological, and neuropsychological tests. ID # Refa Age Sex Neuroimaging studies NCV/EMG/EEG/EP studies Neuropsychological testing 1(74)23M CT normal, possible MRI sinus None reported None reported polyps and mastoid inflammation 2(74)26M None reported None reported None reported 3(74)30F None reported None reported None reported 4(74)56F None reported None reported None reported 5(74)41M None reported None reported None reported 6(71)47M None reported None reported None reported 7(71)33M Abnormal PET scan None reported None reported 8(71)32M None reported None reported None reported 9(71)44M None reported None reported None reported 10 (16) <40 NR None reported Normal NCV and EMG None reported studies 11 (16) <40 NR Normal MRI and PET Normal NCV, EMG, EEG, Verbal memory deficits; returned to normal 2 months postexposure BAEP, and VEP studies 12 (16) <40 NR None reported None reported None reported 13 (16)NRNRNone reported None reported None reported 14 (16) >40 M Normal MRI Normal EEG Attention, memory, psychomotor impairments 15 (75,76)383 M None reported None reported 3/3 Verbal memory deficits (RAVLT); Stroop; Trails B; Grooved Peg- (mean) board; performance improved at follow-up testing (+10–12 weeks) 16 (75,76)384 M None reported None reported 7/7 Verbal memory deficits (RAVLT); Trails B; performance improved (mean) 3 F at follow-up testing 10–12 weeks later 17 (75,76)398 M None reported None reported 9/9 Verbal memory deficits (RAVLT); Stroop; Trails B; Grooved Peg (mean) 1F board; performance improved at follow-up testing 10–12 weeks later 18 (78)4593 M None reported None reported Visual contrast sensitivity (mean) 19 F 19 (72,73,77)40 55 M None reported None reported 13/13 with neurological symptoms had RAVLT scores below the (mean) 11 F 10th percentile Abbreviations: BAEP, brainstem auditory evoked potential; EP, electrophysiologic testing; NCV, nerve conduction velocity; VEP, visual evoked potential. aReference numbers in parentheses correspond to references in text.

group. This patient was reportedly unable to accuracy (Digit Symbol) was impaired as being measured. The animals exposed to remember any of the numbers in a five- well. In contrast, performance on simple Pfiesteria culture extracts were assessed for number sequence and only one of four words attention and concentration (Digit Spans), their performance on a radial-arm maze. in a list immediately after his exposure to constructional praxis (block design), verbal Human exposures to Pfiesteria, in contrast, Pfiesteria. Three months after cessation of fluency (controlled oral word association), have been associated with deficits on tests of exposure to Pfiesteria and 8 days after begin- naming (Visual Naming Test), reading auditory verbal learning and visual contrast ning treatment with cholestyramine, the (Boston Diagnostic Aphasia Exam), visual sensitivity. patient reportedly showed improvement in perceptual abilities (Hooper instrument), This experimental system has been memory function. The specific neuropsycho- remote memory, calculations, and language proposed for two uses: rapid screening of logical tests used to document his improve- functions were stated by the examiners to be extracts from cultures of different strains of ment were not reported by the authors. “within expectation.” Pfiesteria; and establishing dose–response rela- Patient B, in the study reported by Neuropsychological tests were performed tionships between the concentration of toxin- Glasgow et al. (16), showed verbal memory on groups of boaters, sport fishermen, and producing Pfiesteria cells in the culture deficits soon after Pfiesteria exposure; his mem- watermen allegedly exposed to waters con- medium and performance of rats in the radial ory function was reported as normal 2 months taining Pfiesteria (75,77,78). Deficits were arm maze. However, the validity of the assay later. A Maryland waterman examined by seen on the RAVLT (77). Compared with an for these purposes has not been established Bever et al. (76) showed attention, memory, unexposed control group, deficits on the and further research in this area is warranted. and psychomotor impairments on neuro- RAVLT and on the Stroop test were more psychological tests. These authors did not prevalent among persons who were exposed Commentary on Research Findings report the specific tests they used, but these to waters containing Pfiesteria (75). Visual Reviewed tests can be inferred from descriptions pro- contrast sensitivity (VCS) deficits were identi- Human exposure to Pfiesteria appears to be vided by Grattan (75), who was a co-author fied in Pfiesteria-exposed subjects (78–80). associated with effects on functioning of the of the article by Bever et al. (76). Inferences Verbal and motor skills, memory, attention, auditory and visual systems as indicated by concerning the tests used in the evaluation of and spatial reasoning were also assessed in performances on the RAVLT and VCS tests. this patient are indicated in parentheses in the some of these subjects (Table 5). Limited data from performance on other neu- following discussion. Testing indicated prob- Behavioral changes were reported in rats ropsychological tests indicate that other cog- lems with delayed recall of verbal and visual exposed to extracts from Pfiesteria culture nitive domains, including attention and information [Rey Auditory Verbal Learning water (81–83). These behavioral changes sug- executive function, may also be affected. Test (RAVLT) and Visual Retention Test, gest impairment of central nervous system Although frequencies of symptoms and respectively]. Selective and divided attention functioning in these animals. However, the neuropsychological findings are reported in deficits were also seen (Stroop and Trails B, significance of these findings is difficult to these group studies, it is nevertheless difficult respectively). In addition, this patient’s perfor- interpret with regard to previously reported to discern from the published reports what mances on tests of psychomotor speed and changes in human behavior because different impairments were seen in which individuals dexterity (Grooved Pegboard) were impaired. parameters of central nervous system and how those impairments relate to Performance on a task of clerical speed and functioning (e.g., visual vs verbal learning) are Pfiesteria exposure. The subjects in these

654 VOLUME 109 | SUPPLEMENT 5 | October 2001 • Environmental Health Perspectives Pfiesteria: review of the science and research gaps

groups were not characterized as to individual The battery neuropsychological tests used tool for detecting possible congenital color histories of exposure, time elapsed from cessa- by Bever and co-workers (75,76) apparently blindness or retinal problems. tion of exposure to date of testing, or age at did not include a test such as Logical VCS measures the function of retinal the time of testing. In these cases, the infor- Memories, which is designed to elucidate the cells and their pathways to the cortex and mation available from case histories and contribution of attention, working memory, cortical function but does not localize neurological work-ups is insufficient to deter- and auditory information processing to deficits. These findings should be correlated mine the neuropathologic basis of the deficits of the kind reported by these authors. with visual evoked potentials (VEPs), which observed neurocognitive deficits. Several Had their evaluation included such a test, measure conduction of impulses from the reports indicate that the neurological deficits the findings of Bever and co-workers would retinal cells through to the optic cortex. are reversible. The complaints of memory and help to better define the contribution of VEPs are sensitive to demyelinating processes other cognitive problems, together with for- encoding and retrieval deficits to the findings such as multiple sclerosis (MS). The VEPs of mal neuropsychological test results from case on the RAVLT reported by these authors patients with MS are also sensitive to changes studies, suggest that the cognitive distur- and others (76). This test battery also lacked in sine wave grating pattern orientation. This bances develop within hours after exposure a test of vigilance, such as the Continuous finding implies cortical pathology because and may subside spontaneously after cessation Performance Test, which may also have been the receptive field of retinal cells is circular or of exposure but may be exacerbated by stren- sensitive to the attention and concentration oval (86) and therefore these cells do not uous exercise. The few reports of neuro- deficits reported by the patient as difficulty have a significant response preference for one imaging studies and neurophysiological following conversations, driving to familiar orientation or another. The VEPs of patients studies do not indicate a neuropathological places, and managing the finances of his with macular degeneration are slowed but are process; they do suggest that the deficits seen business, and revealed by the formal neu- not sensitive to sine wave grating orientation on neuropsychological tests may be due to ropsychological tests of selective and divided (87), further supporting the hypothesis that reversible disruptions of neurotransmission or attention. cortical pathology underlies the sensitivity to cellular respiration processes. Cholestyramine The Neurobehavioral Evaluation System-2 orientation, as the retina is the site of the has been used to treat cognitive and systemic (NES-2) used by Turf et al. (78) does not lesion in macular degeneration. These find- features of Pfiesteria exposure, but no con- include an auditory verbal learning test and ings indicate that VEPs using sine wave grat- trolled trials have been conducted to support therefore would not be expected to detect ing and visual contrast sensitivity studies can its use for this purpose. deficits in this cognitive domain. The NES-2 be used to localize pathology in the visual also does not include a computerized system of patients exposed to Pfiesteria and Directions for Future Research— counterpart to the VCS test used in studies that localization of the pathology will depend Confirming and Extending Prior Studies reviewed here (78–80). Hudnell et al. (84) on the response to orientation of the grating Rey Auditory Verbal Learning Test. Deficits have published reports indicating that subjects stimulus. have been reported on the RAVLT among with VCS deficits might perform below Other neuropsychological tests. While patients exposed to Pfiesteria (77). The results expectation on subsets of the NES-2, includ- verbal memory and visual contrast sensitivity of the RAVLT should be compared with ing the hand–eye coordination test, when deficits are the reported salient features of scores on the California Verbal Learning Test compared with subjects with intact visual Pfiesteria poisoning in the subjects so far stud- (CVLT) to validate these findings. RAVLT function. The potentially confounding effects ied, deficits may also be seen on tests of atten- and CVLT scores also should be compared of such contrast sensitivity deficits should be tion and executive function, visual memory, with performances on the Digit Span test for taken into consideration in evaluating perfor- psychomotor speed, and personality and affect. auditory memory function, which is one of mance on the NES-2. Improvement in cognitive functioning may be seven tests included in the World Health Visual contrast sensitivity tests. A report seen within 3 months after cessation of expo- Organization (WHO) Neurobehavioral Core by Mergler and Blain (85) suggests that the sure, but whether residual deficits can persist Test Battery (see “Other Neuropsychological Lanthony D-15 or the Farnsworth-Munsell indefinitely has not been determined (16,76). Tests”). If an association is found between 100 Hue tests would be more suitable for Among the persons assessed in this series RAVLT scores and performance on Digit assessing color vision in putatively exposed of reports, memory deficits typically were Span tests administered by clinicians, the patients who are suspected to have visual reported within several days after exposure, results could be compared with the computer- deficits. The Lanthony-D15 (comprising 15 indicating that the first neuropsychological administered oral Digit Spans that will be caps of different colors) is a shorter test, tak- assessment of an exposed subject should be available on the Neurobehavioral Evaluation ing approximately 5 min, and would there- done as soon after the exposure as possible. System-3 (NES-3) once it is fully normed. fore be appropriate for assessing larger groups, Serial testing will document clinical course Because the RAVLT has been so heavily while the Farnsworth-Munsell 100 Hue and recovery and help to predict prognosis emphasized, it is essential that any subject (comprising 85 caps of different colors) after cessation of exposure. evaluated with this test have good hearing. should be used for more detailed assessments The WHO Neurobehavioral Core Test RAVLT, CVLT, and Digit Span scores of individual subjects. By contrast, these Battery (NCTB) assesses central nervous should be compared with brainstem auditory authors suggest that the Ishihara Plates are system function. It is composed of seven tests evoked responses as well as simple auditory not good for assessing acquired color vision that measure simple motor function, short- tests. Findings from these tests should be vali- loss, such as may occur in persons exposed to term memory, eye–hand coordination, affec- dated with other tests of attention and other Pfiesteria, because this test is sensitive only to tive behavior, and psychomotor perception auditory memory, i.e., the Verbal Paired red–green color vision loss and thus may not and speed. The battery includes Digit Span Associate Learning Task and Logical detect blue–yellow color vision loss com- for auditory memory; Santa Ana manual dex- Memories test. Their performance on those monly seen in acquired dyschromatopsia. In terity; Digit Symbol for perceptual motor tests should be carefully evaluated using a addition, acquired dyschromatopsia is speed; the Benton visual retention for visual recognition paradigm to determine if any complex, may involve one or both eyes, and is perception and memory; and Pursuit Aiming deficits identified are in encoding or retrieval age dependent. Therefore, the value of the II for motor steadiness. The sensitivity of this of verbal information. Ishihara Plate test would be as a screening battery for detecting exposure to neurotoxins

Environmental Health Perspectives • VOLUME 109 | SUPPLEMENT 5 | October 2001 655 Samet et al.

such as Pfiesteria is limited because it is too and fine-motor control must be tested to With scant descriptive information brief and does not include more complex substantiate the clinical complaints of ataxia about the neurological consequences avail- tasks of attention and executive function such and dysmetria. Should positive findings able from the case reports, only speculations as the Trail Making Test (Trails B), Paced appear within the first 4–6 hr after exposure, can be offered concerning the possible Serial Auditory Addition, oral arithmetic, or serial testing with any measures used is essen- anatomical sites of action. The diarrhea and the Wisconsin Card Sort. tial so that the time course of effect can be nausea described in the case reports suggest The Boston Extended Neurotoxicologic accurately documented. Persistent effects may an effect upon the autonomic nervous sys- Battery (Clinical) (88) contains omnibus tests emerge over days but the longer the time tem, which would also explain the blurred such as the Wechsler Adult Intelligence Test interval until testing, the greater the likelihood vision reported by some affected persons. Scale—Revised as well as tests selected for that any underlying causal relationship will be The central nervous system effects of ataxia, their sensitivity to deficits in particular cogni- obscured. Of course, a well-characterized bio- dysarthria, and dysmetria would indicate a tive domains. This battery is extensive and logical marker of the exotoxin for use in cerebellar site of effect; the postural instabili- requires a full day to administer but would be experimental studies is sorely needed. ties may arise from vestibular imbalance. If very useful in defining well-studied cases with memory is the most prominent effect, one serial assessments to fully elucidate the clini- Pathological Studies can assume that there are neurotoxic effects cal picture of exposure to Pfiesteria. Pathological findings in fish exposed to on either the hippocampal structures (e.g., Performances on various tests with salient Pfiesteria (33) suggest that a demyelinating layer CA1) or the prefrontal cortex area. auditory components could be compared processes is involved in the clinical neurologi- Central nervous system neurotransmitters, with scores or tests of verbal memory to cal manifestations seen in fish. However, such as glutamate, glycine, and dopamine determine if deficits are in the processing of species differences in metabolism of and cel- may be implicated in the mechanism of all auditory information or in auditory mem- lular responses to the toxin have not been cognitive impairment. ory only. Visual–spatial test performance studied. Therefore, no inferences about the could be compared with visual contrast sensi- role of demyelination in reported human Conclusions tivity scores to further localize possible func- responses to Pfiesteria can be made based on The best-documented instances of adverse tional deficits. The addition of tests sensitive these limited findings in fish. In addition, it is effects in humans are the self-reported cases to attention and executive function will reveal unclear whether the demyelinating process of laboratory workers. The clinical findings contributions that deficits in these domains described in this study affects the peripheral in these cases suggest an association between make to the clinical profile. and central nervous systems in the same fash- exposure to Pfiesteria and the development of An annotated version of this battery could ion. Demyelination could result secondarily neurological symptoms consistent with ner- be developed for research work, based on pre- from axonal injury. More pathological studies vous system dysfunction that may persist vious published reports, and could include are needed to see if the reported observations beyond the acute exposure period, suggesting selected tests of attention and executive func- of fish swimming upside down have an that the toxin possesses significant neurotoxic tion (e.g., Trail Making Tests A and B; Digit anatomical correlate in the balancing mecha- potential. Few additional studies of individ- Spans, oral arithmetic, Paced Auditory Serial nisms, such as the vestibular and cerebellar ual cases and groups that were thoroughly Addition; Stroop Test; Wisconsin Card Sort; systems of fish. Such information may shed evaluated within a short time after exposure Continuous Performance Test); visuopatial light on the complaints of ataxia and disori- have been reported thus far. The inconsisten- and visuomotor function (e.g., finger tapping; entation given by allegedly exposed persons. cies in the clinical approaches and test batter- Digit Symbol; Block Designs; Object Magnetic resonance spectroscopy studies ies used to document the effects of exposure Assembly; Boston Visuospatial Quantitative may be useful for documenting the subtle to Pfiesteria make it difficult to interpret the Battery; Santa Ana Form Board; memory changes in brain chemistry that underlie the significance of the neurological findings. (e.g., California Verbal Learning Test; neuropsychological findings thus far reported. Tests of auditory verbal memory and visual Peterson task, visual reproductions, Logical contrast sensitivity appear to be sensitive to Memories, Verbal Pair Associate Learning, Neurotoxicology the central nervous system effects, but fur- Rey Osterreith Complex Figure); verbal and Because P. piscicida is a dinoflagellate, ther research is needed to substantiate these language function (e.g., Boston Naming Test; inferences about its toxicity can be drawn early findings. A dose–response relationship writing sample, reading comprehension and from the behavioral neurotoxicology of cannot be ascertained at this time. the information and vocabulary subtests from toxins produced by other dinoflagellates. To The lack of neuropathological findings Wechsler); mood and affect (e.g., Profile of cause the effects described in the literature, will not permit speculation on specific Mood States or Minnesota Multiphasic the exotoxin must be quite potent: fish are anatomical sites within the human nervous Personality Inventory). affected within minutes of exposure; system that may be vulnerable to the effects reported effects in humans occurred after of Pfiesteria. The possible mechanisms of Clinical Documentation exposure to what must be minute quantities Pfiesteria neurotoxicity have not been eluci- Further clinical studies should better charac- of the toxin in a contaminated spray of dated. The reports of persistent behavioral terize the temporal course of effects of expo- water. The pharmacology of this exotoxin changes and clinical neuropsychological find- sure, documenting the time relationship from has not yet been elucidated. ings suggest that, with sufficient exposure to exposure to symptom onset and the subse- If the apparent prompt uptake in fish is the toxin, more than an acute disruption of quent course of the clinical symptoms. Formal from the skin as well as the gill, it can be neurotransmission occurs. neuropsychological tests must be done while deduced that the exotoxin is lipophilic. For the patient still has clinical complaints about that reason, the toxin would also be taken up Directions for Future Research memory or emotional disturbances. Selected quickly by the nervous system and would With regard to effects on human health, we brief but highly sensitive test instruments or probably move easily across the blood–brain endorse the following research to address clinical tasks should be done to assess atten- barrier to affect the central nervous system. If data gaps: tion, cognitive tracking, working memory, it is lipophilic, the exotoxin could also affect • Aggressive surveillance should be and visual and auditory memory. Tandem the peripheral myelin. maintained in the coastal states where

656 VOLUME 109 | SUPPLEMENT 5 | October 2001 • Environmental Health Perspectives Pfiesteria: review of the science and research gaps

instances of adverse effects on human to the time relationship with exposure environment and human health, the panel health have been reported. and to track the natural history of any adopted a conceptual framework that extends • Any persons considered to have symptoms adverse effects. from the ecological drivers of the presence and related to exposure to Pfiesteria toxin • Epidemiological studies of persons abundance of Pfiesteria to its effects on fish and should be enrolled in a prospective study considered at high risk as a result of occu- people (Figure 1). The panel’s review was with the objective of characterizing the pation are in progress. These studies repre- structured around this framework and its time course of adverse effects. sent an appropriate step in characterizing research recommendations are directed at key •A standard battery of neuropsychological risk to the population because they address points of uncertainty. tests should be adapted and applied uni- groups considered to have high exposure The panel found that uncertainties still formly to all persons considered to have potential. These studies should be con- remain throughout this framework, possible manifestations of exposure to ducted with sufficient standardization of although laboratory-based studies, coming Pfiesteria toxin. A standardized protocol methods to allow ready cross-comparison now from several laboratories, and the sup- for neurological assessment should be of findings and even pooling. porting field observations, provide firm evi- established as well. dence that Pfiesteria can cause fish kills. It is -A questionnaire such as the Boston Conclusions and Directions more difficult, however, to attribute a partic- Occupational and Environmental for Future Research ular fish kill to Pfiesteria. The experimental Neurology Questionnaire (89) should Research on Pfiesteria has the ultimate purpose evidence is sufficient to meet the modifica- be administered to all persons with of providing evidence that will guide develop- tion of Koch’s postulates that has been possible exposure to Pfiesteria to ascer- ment of strategies to protect the health of estu- applied by this panel and others. The toxin tain concomitant exposures to indus- arine ecosystems and of the people at risk for still remains unidentified and there is even trial chemicals and other factors that exposure to Pfiesteria toxin. Researchers must uncertainty as to the number of toxins pro- could produce similar neurological also use the clinical evidence to guide evalua- duced by the organism. Questions remain as manifestations. tion and management of persons affected by to whether there is a pathognomonic ulcer- - The assessment of effects of exposure Pfiesteria toxin. Recognizing the need for ated lesion of the skin caused by Pfiesteria. should be charted with careful attention research to provide a basis for protection of the Using the fish bioassay, several laboratories

Table 6. Summary of areas for future research on ecophysiology, toxicopathology, and human health effects of Pfiesteria. Topical area Future research Ecophysiology of Establish research-quality cultures of P. piscicida for wide distribution among the scientific community for life cycle, physiological, and ecological study P. piscicida Develop more effective and quicker methods to • distinguish P. piscicida in each of its life cycle stages from look-alike species • quantify the abundance, distribution, seasonality, and toxicity of P. piscicida Conduct laboratory and field investigations, using appropriate controls, to clarify the role of organic versus inorganic nutrients, including those in sewage and in wastes from agroindustrial sources, in stimulating growth and toxicity of P. piscicida, and the role of these and nonnutritional factors, including prey and predatory dynamics, in regulating its in situ growth, population dynamics, and seasonal cycles Identify and chemically characterize the substances produced by fish that stimulate life-stage transformations, growth, and toxin production in P. piscicida Assess the role of environmental signals in controlling toxin-producing capability, life-cycle transformations, and functional phenotype occurrences of P. piscicida; determine the accompanying physiological characteristics of its life cycle stages and population strains Effects on fish and Characterize the role of Aphanomyces spp. in causing skin ulcers observed in wild menhaden organisms and the potential interaction of this fungus with other aquatic Pfiesteria toxin in affecting fish organisms Conduct full tissue pathological evaluations of fish from laboratory bioassays of Pfiesteria and in the wild fish from Pfiesteria-associated fish kills Develop standardized protocol and diagnostic criteria for necropsy and histopathologic examination Compile histopathology and necropsy findings in centralized database shared among laboratories Investigate the physiological and toxicological effects of Pfiesteria toxin exposure in fish, especially • neurophysiologic effects • tissue and organ distribution Methods to detect Develop and preserve a standard panel of ichthyotoxic water samples and/or extracts derived from clonal Pfiesteria cultures Pfiesteria and Promote collaboration among labs developing analytical and purification methods by distributing the standard ichthyotoxic samples or extracts toxin Standardize the fish bioassay process to facilitate interlaboratory reproducibility Develop or refine chromatographic methods for toxin analysis and purification: • conventional diode array UV detection • bioassay methods (cytotoxicity, receptor-binding assay, rapid fish bioassay) to guide method optimization Apply mass spectroscopic detection methods to chromatographic procedures to estimate molecular mass of putative toxins Apply NMR methods to determine structure of purified toxins Produce antibodies and develop immunochemical methods to detect the Pfiesteria organism and its toxin in the field Identify genes associated with toxin biosynthesis using genomic screening methods and analysis of gene expression Adapt PCR and/or FISH methods to devices that can be used in the field to detect toxin-producing phenotypes of Pfiesteria Epidemiological and Maintain aggressive surveillance for possible estuary-associated syndrome in the coastal states where adverse effects have been reported among clinical studies persons possibly exposed to waters containing Pfiesteria Enroll symptomatic persons identified through surveillance in a prospective study of the natural history of adverse effects of exposure Establish a standardized protocol for neurological assessment and apply it to all persons considered to have clinical manifestations of exposure to Pfiesteria toxin; the protocol should include a standard battery of neuropsychological tests adapted for the purpose Using a validated questionnaire, ascertain concomitant exposures to other neurotoxic agents (e.g., industrial chemicals, metals) Conduct repeated neuropsychological and clinical evaluations of symptomatic persons to characterize the clinical course of any adverse neurological effects and establish their time relationship with exposure Standardize methods in epidemiological studies of persons at high risk for occupational exposure to Pfiesteria toxin to allow ready cross-comparison of findings or pooled analyses

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have independently confirmed that the • Our very limited understanding of the (Gasterosteus aculeatus) from the Queen Charlotte Islands, organism can kill fish. Pfiesteria toxin; it has not yet been iso- Canada. Can J Bot 75:1936–1940 (1997). 20. Burkholder JM, Glasgow HB, Hobbs CW. Fish kills linked to a Laboratory and field observations indicate lated and its mechanism of action is a toxic ambush-predator dinoflagellate: distribution and environ- that the organism has a complex life cycle. The matter of speculation. mental conditions. Mar Ecol Prog Ser 124:43–61 (1995). current formulation includes 24 stages, but • The incomplete description of the effects of 21. Litaker RW, Tester PA, Colorni A, Levy MG, Noga EJ. The phylo- genetic relationship of Pfiesteria piscicida, Cryptoperidiniopsoid only a few of the stage transformations have exposure on humans; we have a patchwork Sp. Amyloodinoum ocellatum and a Pfiesteria-like dinoflagellate been fully documented with photographic or of data and little longitudinal information. to other dinoflagellates and apicomplexans. J Phycol videographic techniques, and molecular confir- • Finally, the nature and extent of exposures 35:1379–1389 (1999). 22. Litaker R, Steidinger K, Richardson B. Compilation of Molecular mation of the genetic identity of each stage has that place people at risk but remain Information on PLO’s. Available: http://www.redtide.whoi.edu/ not been published. The certainty of the uncharacterized; we need to determine the pfiesteria/molecular/molecular.html [cited 31 August 2000]. panel’s conclusions with regard to both the life magnitude of the potential threat to per- 23. Landsberg JH, Steidinger KA, Blakesly BA. Fish-killing dinofla- cycle of Pfiesteria and its ability to cause fish sons exposed to toxin-contaminated waters gellates in a tropical aquarium. In: Harmful Marine Algal Blooms (Lassus P, Arzul G, Erard E, Gentien P, Marcaillou C, kills reflects the convergence of evidence from through work or recreational activities. eds). Proceedings of the Sixth International Conference on Toxic both the laboratory and the field. Marine Phytoplankton, October 1993, Nantes, France. Paris: For other key points in the panel’s REFERENCES AND NOTES Lavoisier, Intercept Ltd., 1995;65–70. 24. Pinckney JL, Paerl HW, Haugen E, Tester PA. Responses of phy- conceptual framework see Figure 1; however, toplankton and Pfiesteria-like dinoflagellate zoospores to nutri- 1. Centers for Disease Control and Prevention. Results of the pub- ent enrichment in the Neuse River Estuary, North Carolina, uncertainty remains. The extent and nature lic health response to Pfiesteria workshop—Atlanta, Georgia, USA. Mar Ecol Prog Ser 192:65–78 (2000). of the hazard posed to human health by expo- 29-30 September 1997. Mor Mortal Wkly Rep 46:951–952 25. Glasgow HB, Lewitus AJ, Burkholder JM. Feeding behavior of (1997). sure to Pfiesteria toxin remains unknown. the ichthyotoxic estuarine dinoflagellate, Pfiesteria piscicida, on 2. Centers for Disease Control and Prevention. Notice to Readers. The anecdotal reports of affected scientists amino acids, algal prey, and fish vs. mammalian erythrocytes. Possible estuary-associated syndrome [Letter]. Mor Mortal In: Harmful Algae (Reguera B, Blanco J, Fernandez ML, Wyatt T, and individual case reports of persons exposed Wkly Rep 48:381 (1999). eds). Vigo, Spain:Xunta de Galicia and Intergovernmental 3. Centers for Disease Control and Prevention. Surveillance for at times of fish kills are a basis for concern Oceanographic Commision of UNESCO, 1998;394–397. possible estuary-associated syndrome—six states, 1998-1999. and have appropriately led to a broad pro- 26. Lewitus AJ, Willis BM, Hayes KC, Burkholder JM, Glasgow HB Mor Mortal Wkly Rep 49:372–373 (2000). Jr, Glibert PM, Burke MK. Mixotrophy and nitrogen uptake by gram of epidemiological and clinical research. 4. Evans AS. Causation and Disease: A Chronological Journey. Pfiesteria piscicida (Dinophyceae). J Phycol 35:1430–1437 New York:Plenum Medical Books, 1993. Aside from indicating a potential threat to (1999). 5. Fredricks DN, Relman DA. Sequence-based identification of public health, however, the available clinical 27. Griffith D. Exaggerating environmental health risk: the case of microbial pathogens: a reconsideration of Koch’s postulates. the toxic dinoflagellate Pfiesteria. Hum Organ 58:119–127 and epidemiological data provide neither a Clin Microbiol Rev 9:18–33 (1996). (1999). clear picture of the consequences of exposure 6. Marshall HG, Gordon AS, Seaborn DW, Dyer B, Dunstan WM, 28. Stow CA. Assessing the relationship between Pfiesteria and Seaborn AM. Comparative culture and toxicity studies between to Pfiesteria toxin nor an indication of the estuarine fishkills. Ecosystems 2:237–241 (1999). the toxic dinoflagellate Pfiesteria piscicida and a morphologi- 29. Lewitus AJ, Rublee PA, Mallin MA, Shumway SE. Human magnitude of the problem. A patchwork of cally similar cryptoperidiniopsoid dinoflagellate. J Exp Mar Biol health and environmental impacts from Pfiesteria: a science- approaches has been used to describe symp- Ecol 255(1):51–74 (2000). based rebuttal to Griffith. Hum Organ 58:455–458 (1999). 7. Susser M. Causal Thinking in the Health Sciences; Concepts toms and neuropsychological effects and the 30. Oldach D. Regarding Pfiesteria. Hum Organ 58:459–460 (1999). and Strategies of Epidemiology. New York:Oxford University 31. Rublee PA, Kempton J, Schaefer E, Burkholder JM, Glasgow panel could not find a cohesive picture on Press, 1973. HB, Oldach D. PCR and FISH detection extends the range of reviewing the evidence. Work in this area is 8. Hill AB. The environment and disease: association or causa- Pfiesteria piscicida in estuarine waters. Va J Sci 50:325–335 tion? Proc R Soc Med 58:295–300 (1965). further limited by the need to use surrogates (1999). 9. Department of Health Education and Welfare. Smoking and for exposure, since the presence of toxin can- 32. Burkholder JM, Glasgow HB, Steidinger KA. Stage transforma- Health. Report of the Advisory Committee to the Surgeon tions in the complex life cycle of an ichthyotoxic “ambush- not be directly confirmed and exposure can- General. DHEW Publ no. [PHS] 1103. Washington, DC:U.S. predator” dinoflagellate. In: Harmful Marine Algal Blooms Government Printing Office, 1964. not be quantified. (Lassus P, Arzul G, Erard E, Gentien P, Marcaillou C, eds). 10. Burkholder JM, Noga EJ, Hobbs CH, Glasgow HB. New ‘phan- In its review of the ecological evidence, Proceedings of the Sixth International Conference on Toxic tom’ dinoflagellate is the causative agent of major estuarine Marine Phytoplankton, October 1993, Nantes, France. 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