Receptor binding assay for the detection of paralytic shellfish poisoning toxins: comparison to the mouse bioassay and applicability under regulatory use.

The receptor-binding assay (RBA) method for the detection of paralytic shellfish poisoning (PSP) toxins was evaluated for its overall performance in comparison with the mouse bioassay (MBA). An initial study to evaluate the effects of filtering shellfish extracts prior to running the RBA indicated no significant difference between filtered and unfiltered extracts on the determined saxitoxin (STX) concentrations. Next, we tested the RBA assay on 295 naturally contaminated mussel tissue samples, ranging in concentrations from 320 µg STX equiv. kg-1 to 13,000 µg STX equiv. kg-1 by MBA. An overall trend was observed with the RBA giving higher results (256 µg STX equiv. kg-1 on average) than the MBA; however, at low concentrations (< 500 µg STX equiv. kg-1) the RBA results were marginally lower. A third study was conducted using spiked mussel tissue analysed by three independent laboratories, two of which performed the RBA and one the MBA. This multi-laboratory study again showed the RBA to give higher results than the MBA; however, it also revealed that STX determination was accurate by the RBA, unlike the MBA. To optimise the assay for efficient usage under regulatory practice, three suggestions have been made: the use of an initial screening plate to separate those samples that exceed the alert level; use of rapid PSP test kits in the field and in the laboratory for screening negative samples and for early detection of toxicity; and use of an alternate commercially available porcine membrane in place of the laboratory-prepared rat membrane homogenate. The large number of samples analysed and the diversity of the tests conducted in this study further support the RBA as an affordable rapid method for STX detection that is also free of the routine sacrifice of live animals.

Association of an unusual marine mammal mortality event with Pseudo-nitzschia spp. Blooms along the southern California coastline.

During 2002, 2,239 marine mammals stranded in southern California. This unusual marine mammal stranding event was clustered from April to June and consisted primarily of California sea lions (Zalophus californianus) and long-beaked common dolphins (Delphinus capensis) with severe neurologic signs. Intoxication with domoic acid (DA), a marine neurotoxin produced during seasonal blooms of Pseudo-nitzschia spp., was suspected. Definitively linking harmful algal blooms to large-scale marine mammal mortalities presents a substantial challenge, as does determining the geographic extent, species composition, and potential population impacts of marine mammal die-offs. For this reason, time series cross-correlation analysis was performed to test the temporal correlations of Pseudo-nitzschia blooms with strandings occurring along the southern California coastline. Temporal correlations were identified between strandings and blooms for California sea lions, long-beaked common dolphins, and short-beaked common dolphins (Delphinus delphis). Similar correlations were identified for bottlenose dolphins (Tursiops truncatus) and gray whales (Eschrichtius robustus), but small sample sizes for these species made associations more speculative. The timing of the blooms and strandings of marine mammals suggested that both inshore and offshore foraging species were affected and that marine biotoxin programs should include offshore monitoring sites. In addition, California sea lion-strandings appear to be a very sensitive indicator of DA in the marine environment, and their monitoring should be included in public health surveillance plans.

Receptor binding assay for paralytic shellfish poisoning toxins: optimization and interlaboratory comparison.

A receptor binding assay (RBA) for detection of paralytic shellfish poisoning (PSP) toxins was formatted for use in a high throughput detection system using microplate scintillation counting. The RBA technology was transferred from the National Ocean Service, which uses a Wallac TriLux 1450 MicroBeta microplate scintillation counter, to the California Department of Health Services, which uses a Packard TopCount scintillation counter. Due to differences in the detector arrangement between these 2 counters, markedly different counting efficiencies were exhibited, requiring optimization of the RBA protocol for the TopCount instrument. Precision, accuracy, and sensitivity [limit of detection = 0.2 microg saxitoxin (STX) equiv/100 g shellfish tissue] of the modified protocol were equivalent to those of the original protocol. The RBA robustness and adaptability were demonstrated by an interlaboratory study, in which STX concentrations in shellfish generated by the TopCount were consistent with MicroBeta-derived values. Comparison of STX reference standards obtained from the U.S. Food and Drug Administration and the National Research Council, Canada, showed no observable differences. This study confirms the RBA's value as a rapid, high throughput screen prior to testing by the conventional mouse bioassay (MBA) and its suitability for providing an early warning of increasing PSP toxicity when toxin levels are below the MBA limit of detection.