Fluidic force discrimination assays: a new technology for tetrodotoxin detection.

Tetrodotoxin (TTX) is a low molecular weight (approximately 319 Da) neurotoxin found in a number of animal species, including pufferfish. Protection from toxin tainted food stuffs requires rapid, sensitive, and specific diagnostic tests. An emerging technique for the detection of both proteins and nucleic acids is Fluidic Force Discrimination (FFD) assays. This simple and rapid method typically uses a sandwich immunoassay format labeled with micrometer-diameter beads and has the novel capability of removing nonspecifically attached beads under controlled, fluidic conditions. This technique allows for near real-time, multiplexed analysis at levels of detection that exceed many of the conventional transduction methods (e.g., ELISAs). In addition, the large linear dynamic range afforded by FFD should decrease the need to perform multiple sample dilutions, a common challenge for food testing. By applying FFD assays to an inhibition immunoassay platform specific for TTX and transduction via low magnification microscopy, levels of detection of approximately 15 ng/mL and linear dynamic ranges of 4 to 5 orders of magnitude were achieved. The results from these studies on the first small molecule FFD assay, along with the impact to detection of seafood toxins, will be discussed in this manuscript.

Paralytic shellfish poisoning: seafood safety and human health perspectives.

Paralytic shellfish poisoning (PSP) is the foodborne illness associated with the consumption of seafood products contaminated with the neurotoxins known collectively as saxitoxins (STXs). This family of neurotoxins binds to voltage-gated sodium channels, thereby attenuating action potentials by preventing the passage of sodium ions across the membrane. Symptoms include tingling, numbness, headaches, weakness and difficulty breathing. Medical treatment is to provide respiratory support, without which the prognosis can be fatal. To protect human health, seafood harvesting bans are in effect when toxins exceed a safe action level (typically 80 microg STX eq 100 g(-1) tissue). Though worldwide fatalities have occurred, successful management and monitoring programs have minimized PSP cases and associated deaths. Much is known about the toxin sources, primarily certain dinoflagellate species, and there is extensive information on toxin transfer to traditional vectors - filter-feeding molluscan bivalves. Non-traditional vectors, such as puffer fish and lobster, may also pose a risk. Rapid and reliable detection methods are critical for toxin monitoring in a wide range of matrices, and these methods must be appropriately validated for regulatory purposes. This paper highlights PSP seafood safety concerns, documented human cases, applied detection methods as well as monitoring and management strategies for preventing PSP-contaminated seafood products from entering the food supply.

Comparative determination of paralytic shellfish toxins (PSTs) using five different toxin detection methods in shellfish species collected in the Aleutian Islands, Alaska.

Paralytic shellfish poisoning (PSP), a human illness caused by the ingestion of shellfish contaminated with paralytic shellfish toxins (PSTs), has been reported in Alaska for decades. These poisoning incidents have resulted in losses to local economies due to shellfish harvest closures. Thus the development of an effective biotoxin monitoring program designed specifically for the remote regions of Alaska would provide protection for public health and allow for a viable shellfish industry. The present study provides data useful for the development of an effective toxin screening protocol by comparing PST levels quantified in shellfish by many of the currently available PST detection techniques. Seven bivalve species were collected along beaches of the Aleutian Islands from June 2006 to September 2007. The concentration of PSTs was quantified and compared using five different analytical methods: the mouse bioassay, high performance liquid chromatography (HPLC), receptor-binding assay, the commercially available Jellett Rapid PSP Test strips, and an enzyme linked immunosorbent assay technique. The Association of Official Analytical Chemists (AOAC)-approved HPLC method proved to be valuable for characterizing the suite of individual PSTs in each species for research purposes, but was not considered practical for rapid toxin screening in remote Alaskan regions due to its time-consuming nature and requirement of expensive equipment and considerable expertise. In the present study, Jellett test strips were shown to be an effective tool for rapid screening, however due to the high percentage of false positives, subsequent validation via AOAC-approved methods would be required to prevent unnecessary closures.

Non-traditional vectors for paralytic shellfish poisoning.

Paralytic shellfish poisoning (PSP), due to saxitoxin and related compounds, typically results from the consumption of filter-feeding molluscan shellfish that concentrate toxins from marine dinoflagellates. In addition to these microalgal sources, saxitoxin and related compounds, referred to in this review as STXs, are also produced in freshwater cyanobacteria and have been associated with calcareous red macroalgae. STXs are transferred and bioaccumulate throughout aquatic food webs, and can be vectored to terrestrial biota, including humans. Fisheries closures and human intoxications due to STXs have been documented in several non-traditional (i.e. non-filter-feeding) vectors. These include, but are not limited to, marine gastropods, both carnivorous and grazing, crustacea, and fish that acquire STXs through toxin transfer. Often due to spatial, temporal, or a species disconnection from the primary source of STXs (bloom forming dinoflagellates), monitoring and management of such non-traditional PSP vectors has been challenging. A brief literature review is provided for filter feeding (traditional) and non-filter feeding (non-traditional) vectors of STXs with specific reference to human effects. We include several case studies pertaining to management actions to prevent PSP, as well as food poisoning incidents from STX(s) accumulation in non-traditional PSP vectors.

Saxitoxin puffer fish poisoning in the United States, with the first report of Pyrodinium bahamense as the putative toxin source.

BACKGROUND: From January 2002 to May 2004, 28 puffer fish poisoning (PFP) cases in Florida, New Jersey, Virginia, and New York were linked to the Indian River Lagoon (IRL) in Florida. Saxitoxins (STXs) of unknown source were first identified in fillet remnants from a New Jersey PFP case in 2002. METHODS: We used the standard mouse bioassay (MBA), receptor binding assay (RBA), mouse neuroblastoma cytotoxicity assay (MNCA), Ridascreen ELISA, MIST Alert assay, HPLC, and liquid chromatography-mass spectrometry (LC-MS) to determine the presence of STX, decarbamoyl STX (dc-STX), and N-sulfocarbamoyl (B1) toxin in puffer fish tissues, clonal cultures, and natural bloom samples of Pyrodinium bahamense from the IRL. RESULTS: We found STXs in 516 IRL southern (Sphoeroides nephelus), checkered (Sphoeroides testudineus), and bandtail (Sphoeroides spengleri) puffer fish. During 36 months of monitoring, we detected STXs in skin, muscle, and viscera, with concentrations up to 22,104 microg STX equivalents (eq)/100 g tissue (action level, 80 microg STX eq/100 g tissue) in ovaries. Puffer fish tissues, clonal cultures, and natural bloom samples of P. bahamense from the IRL tested toxic in the MBA, RBA, MNCA, Ridascreen ELISA, and MIST Alert assay and positive for STX, dc-STX, and B1 toxin by HPLC and LC-MS. Skin mucus of IRL southern puffer fish captive for 1-year was highly toxic compared to Florida Gulf coast puffer fish. Therefore, we confirm puffer fish to be a hazardous reservoir of STXs in Florida's marine waters and implicate the dinoflagellate P. bahamense as the putative toxin source. CONCLUSIONS: Associated with fatal paralytic shellfish poisoning (PSP) in the Pacific but not known to be toxic in the western Atlantic, P. bahamense is an emerging public health threat. We propose characterizing this food poisoning syndrome as saxitoxin puffer fish poisoning (SPFP) to distinguish it from PFP, which is traditionally associated with tetrodotoxin, and from PSP caused by STXs in shellfish.