Scientists from the University of Maryland have recently associated exposure to fish kills on the Chesapeake Bay with serious health problems, characterized by neurologic and possible immune dysfunction. Although the causative agent of these fish kills has not been identified, these outbreaks resemble others which have occurred in North Carolina and have been attributed to the toxic dinoflagellate Pfiesteria piscicida. Both fisk kills and human health problems appear to be toxin-mediate; however, the toxin or the mode of action have not been characterized. Isolation, identification and characterization of P. piscicida and related species pose special problems due to their morphological similarity and to the complexity of their life cycles. Current taxonomy is based largely upon ultrastructure examination of cellulose armored plates (thecae) from the vegetative asexual stage. Although controversial, some of the reported greater than 24 life cycle stages of P. piscicida, which exhibits both typical biflagellated as well as ameboid morphologies, place it in the order Dinoamoebales. This classification has not been validated by molecular models of taxonomy, in part, because the expertise and BSL3 containment required for culture, identification and toxicity assays are not widely available. Recent cultures of Maryland isolates suggests that in addition to P. piscicida, closely related species are also present, and that current methodology for their culture can be applied. We propose to apply molecular for their culture can be applied. We propose to apply molecular approaches for identification and classification of these organisms based on a number of gene sequences, including rRNA, mtDNA, microsatellite DNA, and conserved protein-coding sequences such as actin. These sequences can be obtained through the application of PCR and """"""""universal"""""""" primers optimized for the application of dinoflagellate genes. We have successfully applied these techniques for molecular characterization of related protists. This work, in conjunction with subtractive genetics, will provide the basis for species-, toxin-, and stage-specific molecular probes for rapid, sensitive, and accurate identification and quantitation. Studies will be coordinate with other research within the Core Facility for the Culture Toxic Dinoflagellates in collaboration with experts in the field.
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