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Lake Karla, Greece, was dried up in 1962 and its refilling started in 2009. We examined the Cyanobacteria and unicellular eukaryotes found during two fish kill incidents, in March and April 2010, in order to detect possible causative agents. Both microscopic and molecular (16S/18S rRNA gene diversity) identification were applied. Potentially toxic Cyanobacteria included representatives of the Planktothrix and Anabaena groups. Known toxic eukaryotes or parasites related to fish kill events were Prymnesium parvum and Pfiesteria cf. piscicida, the latter being reported in an inland lake for the second time. Other potentially harmful microorganisms, for fish and other aquatic life, included representatives of Fungi, Mesomycetozoa, Alveolata, and Heterokontophyta (stramenopiles). In addition, Euglenophyta, Chlorophyta, and diatoms were represented by species indicative of hypertrophic conditions. The pioneers of L. Karla's plankton during the first months of its water refilling process included species that could cause the two observed fish kill events.

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Based on an analysis of an ongoing scientific-political controversy over the toxicity of a fish-killing microorganism, this paper explores the relationship between responsibility and nonhuman contributions to agency in experimental practices. Research into the insidious effects of the dinoflagellates Pfiesteria piscicida (the fish killer) that thrive in waters over-enriched with nutrients, has received considerable attention by both the media and government agencies concerned with public and environmental health. After nearly two decades of research, the question of whether Pfiesteria can be regarded the 'causative agent' of massive fish kills in the estuaries of the US mid-Atlantic could not be scientifically settled. In contrast to policymakers, who attribute the absence of a scientific consensus to gaps in scientific knowledge and uncertainties regarding the identity and behavior of the potentially toxic dinoflagellates, I propose that an inseparable entanglement of Pfiesteria's identities and their toxic activities challenges conventional notions of causality that seek to establish a connection between independent events in linear time. Building on Karen Barad's framework of agential realism, I argue for a move from epistemological uncertainties to ontological indeterminacies that follow from Pfiesteria's contributions to agency, as the condition for responsible and objective science. In tracking discrepant experimental enactments of Pfiesteria that have been mobilized as evidence for and against their toxicity, I investigate how criteria for what counts as evidence get built into the experimental apparatuses and suggest that the joint possibilities of causality and responsibility vary with the temporalities of the objects enacted. This discussion seeks to highlight a thorough entanglement of epistemic/ontological concerns with the ecological/political relevance of particular experiments. Finally, I introduce a new kind of scientific object that--borrowing from Derrida--I call phantomatic. Phantoms don't emerge as such, but appear as traces and are associated with specific matters of concern.

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Patients exposed to the toxic dinoflagellate Pfiesteria develop an illness characterized by secretory diarrhea, conjunctival irritation, skin lesions, and varying degrees of neurologic manifestations. The anion-exchange resin, cholestyramine has been reported in one small case series to be an effective treatment of severe diarrhea associated with Pfiesteria intoxication. A 54-year-old man traveled to the Dominican Republic where he went swimming in what he describes as "dirty ocean water". Within an hour, he noted a generalized burning and itching of his skin. Later on, he noted pruritic vesicular skin lesions, intense frontal headache, and conjunctivitis. A few days later, he complained of abdominal cramping, nausea, and hourly episodes of watery, non-bloody diarrhea. Due to the constellation of symptoms, Pfiesteria intoxication was suspected. On arrival in the United States, he sought medical care for continued symptoms. Physical examination was remarkable for conjunctival injection, linear vesicular lesions (5 cm in length) over his right ankle and left orbit as well as erythema over foreskin of his penis. Mental status and memory were normal. Laboratory studies revealed an elevated serum creatinine, which eventually normalized, and stool studies were negative for leukocytes, blood, and enteric pathogens. Intense diarrhea persisted until he was started on cholestyramine (4 g PO tid). The diarrhea resolved within 2 hours of starting treatment. The headache was initially treated with narcotic agents but only resolved with IV diphenhydramine (25 mg q 4 h). Cholestyramine and diphenhydramine appear to be effective therapeutic agents for tropical-related diarrhea and headache, respectively.

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Toxicity and its detection in the dinoflagellate fish predators Pfiesteria piscicida and Pfiesteria shumwayae depend on the strain and the use of reliable assays. Two assays, standardized fish bioassays (SFBs) with juvenile fish and fish microassays (FMAs) with larval fish, were compared for their utility to detect toxic Pfiesteria. The comparison included strains with confirmed toxicity, negative controls (noninducible Pfiesteria strains and a related nontoxic cryptoperidiniopsoid dinoflagellate), and P. shumwayae strain CCMP2089, which previously had been reported as nontoxic. SFBs, standardized by using toxic Pfiesteria (coupled with tests confirming Pfiesteria toxin) and conditions conducive to toxicity expression, reliably detected actively toxic Pfiesteria, but FMAs did not. Pfiesteria toxin was found in fish- and algae-fed clonal Pfiesteria cultures, including CCMP2089, but not in controls. In contrast, noninducible Pfiesteria and cryptoperidiniopsoids caused no juvenile fish mortality in SFBs even at high densities, and low larval fish mortality by physical attack in FMAs. Filtrate from toxic strains of Pfiesteria spp. in bacteria-free media was cytotoxic. Toxicity was enhanced by bacteria and other prey, especially live fish. Purified Pfiesteria toxin extract adversely affected mammalian cells as well as fish, and it caused fish death at environmentally relevant cell densities. These data show the importance of testing multiple strains when assessing the potential for toxicity at the genus or species level, using appropriate culturing techniques and assays.

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The ichthyocidal activity of Pfiesteria piscicida dinospores was examined in an aquarium bioassay format by exposing fish to either Pfiesteria-containing environmental sediments or clonal P. piscicida. The presence of Pfiesteria spp. and the complexity of the microbial assemblage in the bioassay were assessed by molecular approaches. Cell-free water from bioassays that yielded significant fish mortality failed to show ichthyocidal activity. Histopathological examination of moribund and dead fish failed to reveal the skin lesions reported elsewhere. Fish larvae within "cages" of variable mesh sizes were killed in those where the pore size exceeded that of Pfiesteria dinospores. In vitro exposure of fish larvae to clonal P. piscicida indicated that fish mortality was directly proportional to the dinospore cell density. Dinospores clustered around the mouth, eyes, and operculi, suggesting that fish health may be affected by their direct interaction with skin, gill epithelia, or mucous surfaces. Molecular fingerprinting revealed the presence of a very diverse microbial community of bacteria, protists, and fungi within bioassay aquaria containing environmental sediments. Some components of the microbial community were identified as potential fish pathogens, preventing the rigorous identification of Pfiesteria spp. as the only cause of fish death. In summary, our results strongly suggest (i) that this aquarium bioassay format, which has been extensively reported in the literature, is unsuitable to accurately assess the ichthyocidal activity of Pfiesteria spp. and (ii) that the ichthyocidal activity of Pfiesteria spp. is mostly due to direct interactions of the zoospores with fish skin and gill epithelia rather than to soluble factors.

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Complete small subunit ribosomal RNA, internal transcribed spacer 1 and 2, 5.8S, and partial large subunit ribosomal RNA gene sequences were generated from multiple isolates of Pfiesteria piscicida. Sequences were derived from isolates that have been shown to be ichthyotoxic as well as isolates that have no history of toxic behavior. All of the sequences generated were identical for the different cultures, and we therefore conclude that differences in toxicity seen between isolates of P. piscicida are linked to factors other than genetic strain variation detectable by ribosomal gene sequence analyses.

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Pfiesteria piscicida and Pfiesteria shumwayae are estuarine dinoflagellates thought to be responsible for massive fish deaths and associated human illnesses in the southeastern United States. These dinoflagellates are described as having a complex life cycle involving flagellated zoospores, cysts, and amoeboid stages. Although no Pfiesteria toxin has been identified, certain strains of these dinoflagellates are thought to produce a water-soluble toxin that can kill fish and cause human illness. Recent reports show no evidence for amoeboid stages and indicate that a much more simplified life cycle exists. In addition, researchers have shown that P. shumwayae only kills fish through direct contact that does not necessarily involve the production of one or more toxins. This review summarizes these and other recent findings with an emphasis on establishing basic facts regarding the toxicity and life history of Pfiesteria dinoflagellates.

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BACKGROUND: Pfiesteria piscicida and look-alike dinoflagellates are collectively known as Pfiesteria complex organisms (PCOs). The purposes of this paper are to evaluate the threat that PCOs pose to human health and to clarify some common misconceptions concerning these organisms. METHODS: References about P piscicida and PCOs were identified and reviewed using MEDLINE, Sea Grant databases, and other sources obtained from various Internet sites. RESULTS: Researchers exposed to aerosols of water containing P piscicida in a laboratory setting exhibited a variety of symptoms, including reversible cognitive impairment and loss of short-term memory. This led to multiple biologic, animal model, and epidemiologic studies that are reviewed and discussed. CONCLUSIONS: Exposure to P piscicida and possibly other PCOs can cause human illness. Until further isolation and purification of toxins from PCOs allows for development of tests to detect these toxins, predicting and documenting toxic PCO fish kills as well as potential PCO-related illness will be difficult.

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Since its identification, the dinoflagellate Pfiesteria piscicida has been implicated in fish kills and fish disease in the southeastern United States. Adverse health effects have been reported in researchers working with the organism and in watermen following exposure to a fish kill in Maryland. A bioactive secretion is postulated as the cause of these effects but has not yet been isolated and chemically characterized. The biology and toxicology of this organism remain the topic of debate and research.

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In connection with the CDC National Conference on Pfiesteria, a multidisciplinary panel evaluated Pfiesteria-related research. The panel set out what was known and what was not known about adverse effects of the organism on estuarine ecology, fish, and human health; assessed the methods used in Pfiesteria research; and offered suggestions to address data gaps. The panel's expertise covered dinoflagellate ecology; fish pathology and toxicology; laboratory measurement of toxins, epidemiology, and neurology. The panel evaluated peer-reviewed and non-peer-reviewed literature available through June 2000 in a systematic conceptual framework that moved from the source of exposure, through exposure research and dose, to human health effects. Substantial uncertainties remain throughout the conceptual framework the panel used to guide its evaluation. Firm evidence demonstrates that Pfiesteria is toxic to fish, but the specific toxin has not been isolated or characterized. Laboratory and field evidence indicate that the organism has a complex 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 surrogate measures of exposure to the toxin are major limitations of this work. To protect public health, the panel suggests that priority be given research that will provide better insight into the effects of Pfiesteria on human health. Key gaps include the identity and mechanism of action of the toxin(s), the incomplete description of effects of exposure in invertebrates, fish, and humans, and the nature and extent of exposures that place people at risk.

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