Screening tests for the rapid detection of diarrhetic shellfish toxins in Washington State.

The illness of three people due to diarrhetic shellfish poisoning (DSP) following their ingestion of recreationally harvested mussels from Sequim Bay State Park in the summer of 2011, resulted in intensified monitoring for diarrhetic shellfish toxins (DSTs) in Washington State. Rapid testing at remote sites was proposed as a means to provide early warning of DST events in order to protect human health and allow growers to test "pre-harvest" shellfish samples, thereby preventing harvest of toxic product that would later be destroyed or recalled. Tissue homogenates from several shellfish species collected from two sites in Sequim Bay, WA in the summer 2012, as well as other sites throughout Puget Sound, were analyzed using three rapid screening methods: a lateral flow antibody-based test strip (Jellett Rapid Test), an enzyme-linked immunosorbent assay (ELISA) and a protein phosphatase 2A inhibition assay (PP2A). The results were compared to the standard regulatory method of liquid chromatography coupled with tandem mass spectroscopy (LC-MS/MS). The Jellett Rapid Test for DSP gave an unacceptable number of false negatives due to incomplete extraction of DSTs using the manufacturer's recommended method while the ELISA antibody had low cross-reactivity with dinophysistoxin-1, the major toxin isomer in shellfish from the region. The PP2A test showed the greatest promise as a screening tool for Washington State shellfish harvesters.

Diarrhetic shellfish toxins and other lipophilic toxins of human health concern in Washington State.

The illness of three people in 2011 after their ingestion of mussels collected from Sequim Bay State Park, Washington State, USA, demonstrated the need to monitor diarrhetic shellfish toxins (DSTs) in Washington State for the protection of human health. Following these cases of diarrhetic shellfish poisoning, monitoring for DSTs in Washington State became formalized in 2012, guided by routine monitoring of Dinophysis species by the SoundToxins program in Puget Sound and the Olympic Region Harmful Algal Bloom (ORHAB) partnership on the outer Washington State coast. Here we show that the DSTs at concentrations above the guidance level of 16 µg okadaic acid (OA) + dinophysistoxins (DTXs)/100 g shellfish tissue were widespread in sentinel mussels throughout Puget Sound in summer 2012 and included harvest closures of California mussel, varnish clam, manila clam and Pacific oyster. Concentrations of toxins in Pacific oyster and manila clam were often at least half those measured in blue mussels at the same site. The primary toxin isomer in shellfish and plankton samples was dinophysistoxin-1 (DTX-1) with D. acuminata as the primary Dinophysis species. Other lipophilic toxins in shellfish were pectenotoxin-2 (PTX-2) and yessotoxin (YTX) with azaspiracid-2 (AZA-2) also measured in phytoplankton samples. Okadaic acid, azaspiracid-1 (AZA-1) and azaspiracid-3 (AZA-3) were all below the levels of detection by liquid chromatography tandem mass spectrometry (LC-MS/MS). A shellfish closure at Ruby Beach, Washington, was the first ever noted on the Washington State Pacific coast due to DSTs. The greater than average Fraser River flow during the summers of 2011 and 2012 may have provided an environment conducive to dinoflagellates and played a role in the prevalence of toxigenic Dinophysis in Puget Sound.

Uptake, tissue distribution and excretion of domoic acid after oral exposure in coho salmon (Oncorhynchus kisutch).

Domoic acid (DA) is a potent neurotoxin naturally produced by some pennate diatom species of the genus Pseudo-nitzschia. It is well known that during harmful algal blooms fish can accumulate DA in the gastrointestinal (GI) tract and act as vectors of the toxin to higher trophic level piscivores, often with severe neurotoxic consequences to the predators. Although neurotoxicity and mass mortality have been observed in vertebrates (i.e. marine mammals and sea birds) feeding on contaminated fish, to date there has been no evidence of neurobehavioral toxicity in the fish vectors themselves. It has been hypothesized that fish may not absorb DA from the digestive tract, thus making them insensitive to dietary consumption of DA. To test this hypothesis, we performed oral gavage exposures followed by a time series of tissue dissections to characterize uptake, depuration, and tissue distribution of DA in fish. Intracoelomic (IC) injection exposures (which bypass the GI tract) were also performed to determine if coho neurons are neurologically susceptible to DA. Excitotoxic symptoms were observed in fish via IC injection at similar toxin levels that have been reported to induce excitotoxic symptoms in intraperitoneal (IP) exposures with mammalian models such as mice, suggesting that fish neurons have a similar sensitivity to DA as other vertebrates. Surprisingly, after oral gavage with ecologically relevant doses of DA, the toxin was detected in plasma collected from the dorsal aorta via a permanent intraarterial catheter within 15 min, yet excitotoxic symptoms were not observed. Additionally, DA was detected in liver, heart, spleen, kidney, muscle, brain and bile. These data indicate that although DA is absorbed from the gut, fish do not exhibit neuroexcitatory effects at maximum ecologically relevant oral doses of DA. Tissue distribution and DA uptake and depuration patterns suggest that a majority of the absorbed toxin is excreted via the kidneys and bile, thereby preventing toxic levels of DA from reaching sensitive nervous tissue. Additionally, greater than 20% of total IC administered DA doses were sequestered in bile within 1h of injection in five symptomatic fish, providing evidence for biliary sequestration of the toxin from blood. Here, we comprehensively describe the uptake, depuration, and tissue distribution patterns of DA and propose that renal and biliary processes may serve as primary routes of toxin clearance in fish.