Development of an immunofluorescence technique for detecting Pfiesteria piscicida

Abstract

While several DNA-based methods have been developed for the putatively toxic dinoflagellate Pfiesteria piscicida Burkholder et Steidinger, an independent detection method such as immunofluorescence can be a useful alternative. In this study, P. piscicida-specific antisera were developed, and an immunofluorescence (IF) procedure was optimized. A total of six antisera were raised using whole cells (WCA) and the insoluble cellular fraction (ICF) as antigens, respectively, and their titer and specificity were examined using dot blot analysis and whole cell IF. Results showed that the two antisera produced from the ICF antigen had a markedly higher titer (1500) than the other four yielded from the WCA (200). In addition, the two ICF-derived antisera exhibited much higher species specificity, showing no cross-reaction with P. shumwayae, Cryptoperidiniopsis sp., Karlodinium micrum, and other more distant algae tested, and very low background for field collected samples. In evaluation of the IF technique using a P. piscicida-specific polymerase chain reaction (PCR) technique, results from both methods generally agreed well for both field samples (from eastern Long Island Sound) spiked with cultured P. piscicida and those containing naturally occurring P. piscicida (from Chesapeake Bay tributaries).