Pfiesteria

Background

Pfiesteria piscicida was discovered inadvertently when cultured fish, tilapia, died after exposure to water collected from the Pamlico River in North Carolina in the late 1980s; it was first identified in the wild from water of the Albemarle-Pamlico estuary in 1991 (Burkholder 1997, Burkholder 1992, Steidinger 1996). Originally called "ambush predator dinoflagellate" to describe its behavior with fish prey (Burkholder 1992, Steidinger 1996), Pfiesteria has been associated with fish ulceration disease and fish kills. In its toxic state, this organism can reportedly cause dermal ulcerations to fish, possibly making them less responsive to environmental stimuli. This invites bacteria, parasites and other predatory organisms to prey on the vulnerable fish (Burkholder 1992. Steidinger 1996, Glasgow 1995). Since this time, researchers have worked to understand this organism by defining its geographic distribution and apparently complex life cycle, as well as the impact P. piscicida may have on the environment and human health. Most of the research concerning P. piscicida is still in its infancy, with a high the level of uncertainty surrounding the organism, its life cycle and biology, and its possible toxin(s) production.

One important issue surrounding the research of Pfiesteria concerns the problem and difficulty in identifying the organism. Since the discovery of P. piscicida, other dinoflagellates have been found which are morphologically similar, called "Pfiesteria-like organisms (PLOs)" or "morphylogically-related organims (MROs)." The organisms must be stripped of an outer membrane and viewed under electron microscope in order to accurately identify and distinguish between the various dinoflagellates (Steidinger 1996). The difficulty in organism identification, which cannot be performed in the field, creates uncertainty with respect to earlier research and sampling results (Smith 1998, Steidinger 1996, Fleming 1995).

Life Cycle

The life cycle of Pfiesteria piscicida is reportedly extremely complicated, having 24 different unicellular stages of which the flagellated and amoeboid forms are the most significant. While dormant, Pfiesteria is an encysted amoeba, and is considered not harmful. Once the appropriate conditions are met, described below, the surrounding lipid soluble cyst is dropped, and the emerging cell can potentially cause problems. The most toxic is the "vegetative" stage that seems to be triggered by and multiplies in the presence of fish (Burkholder 1997, Burkholder 1992, Glasgow 1995, Burkholder 1995).

Distribution

Pfiesteria appears to be most abundant in estuaries, regions where a river meets the ocean and the waters combine to create distinct ecosystem (National Estuary Program 1998, Lewitus 1995). This marine environment contains relatively warm, brackish water with elevated nutrient levels and little wave action (Glasgow 1995, Burkholder 1995, Burkholder 1997). Most of the affected fish have been found in calm, shallow, and poorly flushed areas, characteristic of estuarine environments (Anon 1998).

First linked with a massive fish kill in North Carolina in 1991, Pfiesteria has been found on the East Coast of the United States from Delaware to Virginia. This organism has been associated with fish kills in the Chesapeake Bay and other estuarine/river systems in Maryland. One of the key issues surrounding P. piscicida is the presence of other organisms during fish events. Other dinoflagellates including the Pfiesteria-like organisms may be competing or co-causal factors of the fish kills. It is difficult to distinguish between many of these due to the complex identification procedure described above to properly identify each organism (Burkholder 1997, Steidinger 1999, Glasgow 1995, Steidinger 1996, Burkholder 1995).

Effects on Fish

Since its discovery, Pfiesteria has been implicated in various fish-related events over the past decade, yet there have been fish kills and lesioned fish present in estuarine waters for many years, often lasting many weeks. In the laboratory, fish kills apparently caused by Pfiesteria are short-lived; the toxic effects last only for a few hours, after which the organism allegedly reverts to its non-toxic state. The level of toxicity is apparently measured as the number of P. piscicida in the "toxic" amoeba stage (Burkholder 1997, Glasgow 1995, Burkholder 1995, EPA 1998). In a laboratory that attempted to simulate the conditions of the estuarine environments, the effects of Pfiesteria on fish have been tested. Once the fish were removed from experimental aquariums, the levels of Pfiesteria in the alleged toxic state declined. In the areas with fish kills that have tested positive for Pfiesteria, once the fish are removed, there appeared to be a loss of toxicity (ie. number of P. piscicida) within 24 hours (Burkholder 1997, Steidinger 1996).

Ulcerated Disease Syndrome (UDS) has been noted in fish since the 1970s; UDS presents with fish lesions through the deterioration of fish skin. With the discovery of the new marine organism, UDS has now been associated with P. piscicida and other Pfiesteria-like organisms. It is suspected that fish secretions (such as excrement) stimulate the growth of the dinoflagellates, causing a bloom. ). Fish have been reportedly noted to develop other symptoms due to Pfiesteria such as decrease in feeding habits and a loss of instinctive response to predators and danger (Glasgow 1995, Burkholder 1995, Burkholder 1995, Burkholder 1997). It is unclear whether P. piscicida and/or the PLOs actually cause the fish lesions, or if they cause lethargy among fish allowing for other opportunistic microorganisms such as bacteria and/or fungi to prey upon the susceptible fish. In addition to problems with the identification of the organism(s) and toxin(s), there is uncertainty concerning the temporal factors, what causes these lesions specifically, and what triggers each event (Steidinger 1999).

Environmental Conditions

There are several conditions that are suspected to be conducive for the proliferation of the toxic form of P. piscicida. However, it remains uncertain what actually causes the dinoflagellate to change from its non-toxic stage, triggering the apparent toxic behavior and "attack" on fish (Anon 1998, Burkholder 1997).

Fish kills have occurred frequently along the East Coast of the US for centuries. The majority of fish kills during the 1980s were attributed to low amounts of dissolved oxygen. Due to limited levels of available oxygen and increased nutrient composition, large numbers of fish died as a result of incompatible living conditions. Other possible explanations include changes in the chemical balance of the marine environment that influence the microscopic community. Nitrogen and phosphorus are two of the most important nutrients, and the shifting ratio of the two alters the composition of coastal waters. Phosphorus has been increasing, and there is a possible hypothetical direct relationship between the amount of both phosphorus and dinoflagellates. As a result, Pfiesteria is alleged to thrive in the excess of this element. However, the true relationship between these elemental factors and the presence of Pfiesteria has not been causally defined (Anon 1998, Burkholder 1997, Steidinger 1999, Glasgow 1995).

One of the popular explanations of the latest surge in dinoflagellates in general and Pfiesteria itself is the effects of humans on the environment. Human activities may be responsible for elevated bacterial counts due to wastewater solid effluent from agricultural facilities, wastewater treatment plants, and other high nutrient activities. These bacteria serve as a vital food source for the dinoflagellates and fish that in turn are eaten by P. piscicida, promoting rapid growth. Although these problems are believed to be factors, there has been no substantiated evidence that reducing the phosphorus count and/or nutrient load will conclusively eliminate or even decrease the fish events and level of Pfiesteria in the waters (Anon 1998, Steidinger 1999).

Clinical Presentation

There have been several studies investigating the possible health effects of Pfiesteria exposure on humans. These studies have focused on people who were potentially exposed occupationally and recreationally during events and/or laboratory work predominantly in North Carolina and Maryland.

When evaluating the epidemiologic research in this area, there are a several issues of epidemiological concern (Anon 1998, Fleming 1995). The basic science research necessary to study these organisms is just beginning. At present, there exists no easily available method of identifying the Pfiesteria piscicida organism and the Pfiesteria-like organisms, especially in the field. Furthermore, as discussed above, the presumed toxin(s) associated with these organisms have not been identified. Therefore, there are presently no useful measures of exposure for epidemiologic or clinical studies. Instead, exposure has been defined as self-reported occupational and/or recreational exposure to estuarine waters by skin and/or aerosol route (CDC 1997). Furthermore, in part hampered by the lack of exposure measures, the measures of possible human health effects have tended to be predominantly self-report of a wide variety of symptoms without objective evidence of effect (CDC 1997, Smith 1998, Fleming 1995). Without appropriate exposure and effect measures, it is difficult to accurately define the possible human health effects associated with exposure to these organisms. Therefore, the epidemiologic research of the Pfiesteria piscicida organism and the Pfiesteria-like organisms is its infancy and should be interpreted as such.

Initial Human Case Reports

A case series of 10 individuals (reported by these same individuals) working in various laboratory settings associated with Pfiesteria research was analyzed for illness due to possible aerosolized and dermal exposure aquarium water where Pfiesteria organisms were being studied (Glasgow 1995). Cultures of the organism were grown to mimic natural water conditions to determine basic information regarding life and reproductive cycles. Exposure to this organism and the presumed aerosolized toxins were defined as working with cultures of Pfiesteria for 1-2 hours over several months, although two of the individuals were reportedly exposed by occupying offices near the culture area (Glasgow 1995).

The laboratory workers reported multiple and varied symptoms (including sensory disturbances, stomach cramping, memory loss, respiratory irritation, emotional lability, etc.) for varying degrees of time associated with occupational exposure to the aquarium water and its aerosol. The symptoms, especially the short term memory loss, reportedly lasted for weeks to months; in general, elimination of exposure resulted in the resolution of the symptoms. The investigators associated the degree of exposure with the length of time the individual was symptomatic, particularly the memory loss. Of note, two persons reported symptom resolution after using face masks/ventilators. One person was believed to be "modeling" since symptoms were only reported in the presence of another individual with reporting eye irritation. Unfortunately, systematic testing was not carried out on these individuals, and their improvement is based solely on anecdotal report.

Considerable but varied medical evaluation was carried out on a subgroup of the workers, including neurologic examination, routine blood hematology and chemistries, a toxicology screen for heavy metals, EMG/NCV, spinal fluid evaluation, brainstem and visual evoked potentials, MRI, PET scan, and neuropsychologic testing. The majority of these tests were within normal limits or inconclusive. Only one person apparently received neuropsychologic testing, with results reported as a definite organic syndrome with amnestic syndrome involving verbal more than visual disturbances (Burkholder 1997, Glasgow 1995).

The reported memory loss and other neuropsychological symptoms from possible aerosolized and dermal exposures to Pfiesteria organisms were taken seriously by members of the scientific community because of prior studies documenting irreversible memory loss among individuals exposed to another marine toxin, domoic acid (Perl 1990). In addition, aerosolized brevetoxin exposure from the Florida Red Tide had been associated with reported human health effects (Fleming 1998). The Glasgow et al (1995) case series represents the first reported cases of possible human health effects from apparent occupational exposure to Pfiesteria organisms. No a priori strict case definition with objective health effects criteria was applied to determine the cases and the non-cases. The people who were cases were categorized based on their self-report of symptoms and work exposure. Exposure itself was loosely defined based on job title and physical location; there was no objective measure of exposure, such as levels of Pfiesteria or toxin(s). The entire denominator of potentially exposed persons was not evaluated. Thus, the case definition consisted of an extremely broad definition of symptoms and possibly associated diseases, without consistent objective measures and with a vague exposure definition.

Further Case Series

A prolonged fish kill with fish with lesions occurred in the Chesapeake Bay and Pocomoke River estuary along the eastern shore of Maryland, starting in the Fall of 1996 and augmenting through the summer of 1997. During the summer of 1997, Pfiesteria organisms and Pfiesteria-like organisms were reported by Burkholder and her colleagues in water sample analyses (Grattan 1998). This resulted in a flurry of human health research activity, as well as the temporary closure of the Maryland waterways to commercial activity.

A case series was reported by a local physician (Shoemaker 1998, Shoemaker 1997). These patients reported experiencing several different symptoms such as rashes, headaches, diarrhea, and memory loss. Some of the cases had skin lesions or diarrhea only; others burning sensations and/or memory loss; while still others reported a variety of symptoms including those mentioned, in addition to dizziness, nausea and shortness of breath. The onset of these symptoms ranged from a few days to months after the fish kills. Reportedly psychometric testing done on an unspecified subset of patients revealed the "fingerprint of abnormalities seen with Pfiesteria exposure." Although labeled a "syndrome," there was no consistent symptom complex apparent to link all of these patients together. There were no objective measures of exposure. The cases reported recreational and/or occupational exposure to river water in the area of the fish kills or to killed fish. Allegedly the cholesterol binding agent, cholestyramine, provided relief from symptoms in a subset of cases; however, the drug was administered in a non-blinded, non-random fashion (Shoemaker 1998, Shoemaker 1997).

A group of Maryland investigators collaborated to evaluate individuals with predominantly occupational exposure to estuarine waters with fish kills in the Chesapeake Bay status (Grattan 1998). Initially 13 persons were examined, but ultimately the study included 24 participants with variable exposure primarily through their occupation in large fish kills in August of 1997 and earlier. In this study, participants were asked for information regarding medical history, symptoms, and 13 were given a medical evaluation and lab analysis. A battery of neuropsychological tests was given to determine effects in the individuals. These 24 people were compared to a control group of reportedly similar age, sex, education and occupation; this control group was assembled after the original evaluation. Alleged human health effects were evaluated by exploring cases (complaining of symptoms) who ranged from lab workers to fisherman, all of whom were involved in some aspect of the clean-up and investigation of the fish kills. Analysis of possible human health effects in the study population (5 excluded, 19 remaining) was based on neuropsychological tests that measured learning and memory capabilities (Rey AVLT), attention (Stroop Color-Word Test, Trails), and motor skills (Grooved Pegboard task), in addition to lab tests, dermatological exams, and other neuropsychological tests. The medical examination of the subgroup included pulmonary function tests and skin biopsies if necessary, and laboratory tests (Grattan 1998)

The exposure definition was based on the reported activities the individual participated in during a fish kill with presumed exposure to Pfiesteria as a surrogate for exposure to the alleged toxin(s). The possible routes of exposure were through variable inhalation and/or skin contact with contaminated water. There were varied reported estuarine water exposures, ranging from "high" (defined as 6-8 hours/day) to "moderate" (8-20 hours/week) to "low" with little or no direct skin contact (less than 40 hours total); the job categories ranged from "watermen" to government environmental workers. The highly exposed group consisted of 7 out of 8 watermen who worked in the effected estuaries. Reportedly the majority of the participants were evaluated within 2 weeks from their last exposure to estuarine waters. Two of the 24 exposed subjects were excluded due to incomplete symptom data.

The investigators reported that the cases were more likely to complain of neurological problems than the controls, although there was no definitive difference between the exposed and controls groups with either the medical or lab exams. The exposed group was noted to have lower scores on the some of the neuropsychological exams compared to the controls. There was a reported difference in one of five parts of the learning tests between the four groups, with the highly exposed group reportedly learning significantly fewer words. A linear response was noted, indicating that subjects with the reported highest exposure had the lowest performance on the Stroop Color-Word Test, AVLT, and Grooved Pegboard task (Grattan 1998). investigators were not blinded to the exposure status of all participants, although statistical analysis reportedly found no difference.

During follow-up examinations, researchers found the test scores on part of the Rey AVLT improved, interpreted by the investigators as a reversal of the effects of the possible toxic exposure. There were no differences among the other exams, including dermatologic. The researchers concluded that the effects of Pfiesteria and the presumed toxins can be eliminated by removing the exposure (Grattan 1998). Of note, the follow-up exams were only performed on 10 of the original exposed participants and 12 of the original control group individuals in the study.

There are a number of epidemiologic concerns raised by this study. The original study group was a case series of individuals self-defined in terms of their exposure and health effects labeled "cases" and "controls"; the additional cases and "control group" were assembled later. No apriori hypotheses were formulated. In addition, no objective measures of exposure other than self-report of estuarine water contact were used to categorize subjects into the study subpopulations. The actual study numbers are very small, especially with the exclusion of cases and the subsequent incomplete follow up; therefore, the power of the study to detect a true significant difference is very small. This is especially true when there is the application of multiple statistical tests as is necessary when analyzing the large battery of neuropsychological tests administered by the investigators. Further concerns intrinsic to neuropsychological testing include significant effects of confounding from education and socio-economic class, especially in the setting of significant anxiety and lack of baseline information for different population subgroups (Smith 1998, Rullan 1998). Given these limitations, Grattan et al (1998a) study is limited in its generalizability and should be viewed as a hypothesis-generating pilot study (Grattan 1998).

Other human health effect related studies

Many of the same Maryland investigators published a series of articles directed at physicians on the possible human health effects and other aspects of Pfiesteria (Morris 1998). In general, these articles represent further publication of the already discussed articles by Grattan et al (1998a), Burkholder et al (1997) and Glasgow et al (1995) (Burkholder 1997, Glasgow 1995, Grattan 1998). One study by Golub et al (1998) attempted to use participants in a toll free hotline to evaluate whether consumption of seafood was associated with self-reported Pfiesteria exposure and a low score on the neuropsychological test described above, the Rey Auditory Verbal Learning Test (Golub 1998). Reportedly, although associated with fishing and handling fish with lesions, a decreased score was not associated with seafood consumption.

As a part of a North Carolina study, Hudnell (1998) investigated the association between fish kills reportedly caused by Pfiesteria piscicida and the effects on vision (as another neurologic indicator) (Hudnell 1998). Twenty-two people were studied in the "exposed" group, defined as individuals who worked in estuaries where Pfiesteria was present. The "unexposed" group, matched for age, gender, and education, although not for occupation, consisted of 20 individuals who worked offshore but lived in estuarine areas. The vision testing involved a visual acuity test for normalization and the Visual Contrast Sensitivity (VCS) test. The VCS analyzes the ability to distinguish between light and dark patterns; VCS has been used to detect neurotoxic effects in adults with other exposures such as solvents. These tests were administered to both groups during the battery of tests in a larger case series study. Hudnell found that there was no significant difference between the two groups for the visual acuity test, however there was reportedly a difference in the VCS test indicating that the "exposed" group exhibited lower scores in pattern detection. Further analysis excluding subjects of both groups with a history of neuropsychological impairment and excluding those without direct fish kill exposure continued to show a VCS deficit. As with Grattan et al (1998a), although possibly hypothesis generating, this study cannot be considered conclusive of neuropsychological deficits associated with Pfiesteria exposure (Grattan 1998). In particular, there is a lack of objective exposure measurement (with possible exposure misclassification) and unexposed baselines, as well as statistical issues associated with multiple tests with a small study population.

Diagnosis

In September 1997, the Centers for Disease Control and Prevention (CDC) held a "Public Health Response to Pfiesteria Workshop" in order to coordinate response teams for states affected by these and other estuarine organisms (CDC 1997). This meeting and the resulting definitions were created jointly in an effort to bring uniformity to the research and evaluation of fish events. Definitions for environmental exposure to Pfiesteria or morphologically related organisms (MROs) (also known as "Pfiesteria-like"), with subsequent modifications over time, are as follows:

EXPOSURE DEFINITION

The cluster of human health effects that have been defined as signs and symptoms persisting at least 2 weeks or more (except for skin rash or burning) with exposure to Pfiesteria or MROs are:

SIGNS AND SYMPTOMS DEFINITION

Given the high degree of exposure uncertainty, the above disease and exposure description was labeled "Estuarine Associated Syndrome (EAS)." Under the direction and funding of the CDC, several states began investigating the human health effects of Pfiesteria and other estuarine organisms using EAS as a surveillance tool.

Due to the lack of identification of a specific organism, the similarity of Pfiesteria to other dinoflagellates (PLOs and MROs), the lack of resources to accurately sample alleged exposures, and the absence of identified toxin(s), the CDC/States definition of a case of EAS was further broadened to encompass all individuals who are exposed in estuaries with similar symptoms. Researchers established "Possible Estuarine Associated Syndrome (PEAS)" as the name for the illness (with the same qualifying symptoms as EAS) reported after exposure to any estuarine environments, even those without the confirmed presence of Pfiesteria or MROs. Such ambiguity reflects the uncertainty that the fish events and reported human health effects are due to Pfiesteria alone, other dinoflagellates, or something else entirely.

As per the CDC/States protocol for PEAS/EAS surveillance, the PEAS cases undergo an initial interview to see if they meet the symptom and exposure criteria. For the purposes of exposure, the criteria become less stringent. The individual must only report having been exposed to an estuary (regardless of presence of fish kills, lesions or Pfiesteria), in addition to appropriate symptoms. For example in Florida, the South Florida Poison Control Center and a medical response team from the University of Miami are investigating individuals suspected of having both exposure and symptoms that are reportedly associated with exposure to Florida estuarine waters. In addition, efforts were begun in several mid-Atlantic states to assemble "cohorts" (really non-randomly selected subpopulations) of possibly estuarine and HAB exposed individuals to monitor for possibly human health effects over time.

Management and Treatment

To date, there is a small body of suggestive but necessarily inconclusive scientific research in the area of possible human health effects resulting from exposure to Pfiesteria and the Pfiesteria-like organisms. As has been stated above, without objective and easily applicable measures of exposure, as well as a lack of identifiable toxin(s), it is very difficult to perform definitive epidemiologic studies of the possible human health effects of Pfiesteria and the Pfiesteria-like organisms. This situation is further complicated by the apparent necessity of using batteries of neuropsychologic tests as the objective measure of human health effect, as well as self-report of a large range of symptoms (as seen in the P/EAS case definition). Neuropsychologic testing is best performed on relatively large populations with pre-exposure baselines and/or with carefully matched controls in terms of education, socio-economic class, and other possible confounders such as alcohol exposure.

Therefore, at this time, there are no known management and treatment for any diseases that might be associated with Pfiesteria and the Pfiesteria-like organisms.

Chemical Structure

No toxins have been identified to date for Pfiesteria and the Pfiesteria-like Organisms.

Molecular Mechanism of Action

As stated earlier, no toxin(s) associated with Pfiesteria have been isolated. In a series of small experiments, Levin et al (1997) injected albino Sprague-Dawley rats subcutaniously with aquarium water provided by Burkholder's laboratory (Levin 1997). Reportedly recently collected samples (transported frozen) were associated with learning impairment, specifically for learning new tasks, as well as deficits in habituation of arousal and rearing behavior in a functional battery, lasting up to 10 weeks from injection. A single observer was reportedly blind to exposure status. No other effects were noted on the hematologic or pathologic examinations. There is no characterization of the injected aquarium water beyond ammonia and nitrate levels with Pfiesteria cell counts from reportedly fish-lethal cultures. The small sample size, issue of mixed substances in the injections, and mixed results of the statistical testing make this study inconclusive but hypothesis generating.

Research is underway which seeks to identify toxin(s) (as well as what may stimulate toxic transformation) that may be produced by this dinoflagellate. The discovery of toxin(s) would provide the necessary causal link between the fish events and the organism. The research community is reportedly searching for at least three toxins: two lipid-soluble and one water-soluble, to date without success. One of the lipid-soluble toxins (dermonecrotic) may cause fish ulcerations, the other one is labeled the lipid-soluble lethal factor (LSLF); the water-soluble toxin may have possible mechanisms involving the presumed neurotoxic effects on humans (Anon 1998). Discovery of the toxin(s) could lead to necessary dose-response relationships and biological mechanisms needed to determine possible human health effects. Without the definitive presence of a toxin, much of the reported effects can only remain suspect until validation and discovery of a specific marine toxin is proven to be produced by Pfiesteria. Furthermore, evaluation of possible human health effects is hampered by the lack of identifiable toxin and biomarkers of exposure (Anon 1998, Steidinger 1999, Fleming 1995).

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