Finding Pfiesteria Fast.

Science is slowly gaining ground on the still-mysterious Pfiesteria, a toxic dinoflagellate blamed for human illness and massive fish kills in two U.S. states during the past decade. Aiding researchers are recent breakthroughs that let them identify Pfiesteria more quickly and accurately.

Parke A. Rublee, a professor of biology at the University of North Carolina at Greensboro, working with JoAnn M. Burkholder, a professor of marine science at North Carolina State University, first developed rapid techniques to identify Pfiesteria. Their polymerase chain reaction (PCR) assay, available in 1998, is used in monitoring efforts by several states. Recently, a team led by David W. Oldach, an assistant professor of medicine at the University of Maryland School of Medicine in Baltimore, used a technique known as the heteroduplex mobility assay (HMA) to identify ribosomal gene sequences from Pfiesteria species. With that information, they developed other PCR assays for Pfiesteria species that are nearly 100% accurate in identifying their presence in water and sediment samples.

Pfiesteria was first identified in 1988 but didn't draw much attention until it was tagged as the culprit in a series of fish die-offs throughout the 1990s that killed over one billion fish, mostly in North Carolina but also in Maryland. Pfiesteria also is a suspect in fish kills elsewhere along the Eastern seaboard. Some people who worked near or with water or sediment in fish kill areas have suffered short- and long-term symptoms believed to be linked to Pfiesteria such as memory loss, learning impairment, breathing difficulty, nausea, lethargy, and skin lesions. (These symptoms match health problems sustained by laboratory staff working with toxic Pfiesteria cultures until such efforts were restricted to biohazard III containment facilities.)

Before the breakthroughs with HMA and PCR, investigators needed nearly two weeks using a scanning electron microscope and thecal plate analysis to confirm whether Pfiesteria was in a water sample. With improved PCR, that time has been slashed to as little as two hours of lab time, and even faster equipment that can work in the lab or the field is coming on the market. If other toxic algae are believed to be present, HMA (which takes about 48 hours) can then be used to assess which species are present in the water sample, and novel HMA patterns representing other organisms can then be targeted for sequencing analyses. Though the technique allows rapid identification of Pfiesteria, it isn't foolproof; still, public health officials are better equipped to decide quickly if they need to warn water users of a possible hazard.

To develop the technique, Oldach's team gathered water samples from an outbreak area, then filtered them to extract material suspected to contain Pfiesteria. Together with Rublee and Burkholder, they used PCR assay to amplify a region of the microbes' DNA, as described in the 11 April 2000 issue of Proceedings of the National Academy of Sciences. Using a special gel apparatus, they then ran an HMA, which works off the fact that DNA heteroduplexes--nucleic acid molecules composed of two chains, with each derived from a different parent molecule-migrate more slowly through the gel than homoduplexes. With HMA, a "signature" sequence pattern was identified that was present in all of the toxic Pfiesteria cultures assessed, as well as in water samples collected during Pfiesteria-related fish kills.

Through this approach, the investigators were able to overcome the problem that pure Pfiesteria cultures were not yet available for DNA sequencing procedures, and that some cultures (particularly those in which toxicity was maintained through the regular introduction of live fish prey) are contaminated by other eukaryotic organisms. Once the HMA-generated partial sequence information was available, the investigators were able to complete ribosomal gene sequencing work for these and related organisms, and to develop highly specific PCR assays for P. piscicida and P. shumwayae sp. nov., as reported in the November 2000 issue of Applied and Environmental Microbiology.

But HMA and PCR still can't determine whether Pfiesteria species are in a toxic stage. Burkholder has shown that these dinoflagellates morph through many stages--only some of which are toxic to humans or fish--during their complex life cycles. A toxic phase can develop and vanish in a matter of hours--or weeks. Only the fish bioassay, an expensive, multistep procedure, can tell whether Pfiesteria from estuarine waters is in actively toxic mode. HMA and PCR also can't pinpoint how many Pfiesteria cells are in the water or sediment. (It takes about 100 actively toxic Pfiesteria cells per milliliter of water to cause fish health problems, and about 300 cells per milliliter to kill fish.) And researchers are still trying to identify the actual toxin and triggering mechanisms involved in Pfiesteria toxicity. Until these factors are identified, monitoring for the two species alone will provide only a few pieces of the puzzle needed for confirmation of a toxic Pfiesteria event.