Brevetoxin in blood, biological fluids, and tissues of sea turtles naturally exposed to Karenia brevis blooms in central west Florida.

In 2005 and 2006, the central west Florida coast experienced two intense Karenia brevis red tide events lasting from February 2005 through December 2005 and August 2006 through December 2006. Strandings of sea turtles were increased in the study area with 318 turtles (n = 174, 2005; n = 144, 2006) stranding between 1 January 2005 and 31 December 2006 compared to the 12-yr average of 43 +/- 23 turtles. Live turtles (n = 61) admitted for rehabilitation showed clinical signs including unresponsiveness, paresis, and circling. Testing of biological fluids and tissues for the presence of brevetoxin activity by enzyme-linked immunosorbent assay found toxin present in 93% (52 of 56) of live stranded sea turtles, and 98% (42 of 43) of dead stranded sea turtles tested. Serial plasma samples were taken from several live sea turtles during rehabilitation and toxin was cleared from the blood within 5-80 days postadmit depending upon the species tested. Among dead animals the highest brevetoxin levels were found in feces, stomach contents, and liver. The lack of significant pathological findings in the majority of animals necropsied supports toxin-related mortality.

Brevetoxicosis in seabirds naturally exposed to Karenia brevis blooms along the central west coast of Florida.

Harmful algal bloom events caused by the dinoflagellate Karenia brevis occurred along the central west Florida, USA, coast from February 2005 through December 2005 and from August 2006 through December 2006. During these events, from 4 February 2005 through 28 November 2006, live, debilitated seabirds admitted for rehabilitation showed clinical signs that included disorientation, inability to stand, ataxia, and seizures. Testing of blood, biologic fluids, and tissues for brevetoxin by enzyme-linked immunosorbent assay found toxin present in 69% (n=95) of rehabilitating seabirds. Twelve of the 19 species of birds had evidence of brevetoxin exposure. Commonly affected species included Double-crested Cormorants (Phalacrocorax auritus), Brown Pelicans (Pelecanus occidentalis), Great Blue Herons (Ardea herodias), and Common Loons (Gavia immer). Serial blood and fecal samples taken from several live seabirds during rehabilitation showed that brevetoxin was cleared within 5-10 days after being admitted to the rehabilitation facility, depending on the species tested. Among seabirds that died or were euthanized, the highest brevetoxin concentrations were found in bile, stomach contents, and liver. Most dead birds had no significant pathologic findings at necropsy, thereby supporting brevetoxin-related mortality.

Maternal-fetal transfer of domoic acid in rats at two gestational time points.

BACKGROUND AND OBJECTIVES: Prenatal exposure to asymptomatic doses of domoic acid (DA) causes learning and memory deficits later in life; therefore, we sought to measure distribution of DA in maternal plasma and brain, prenatal brain, and amniotic fluid 1 hr after exposure, a time frame that normally encompasses acute seizure behavior. METHODS: Pregnant rats were given a single intravenous dose of DA (0.6 or 1.6 mg/kg body weight) at either gestational day (GD) 13 or GD20, which correspond to the beginning of rat embryo neurogenesis and the last day of gestation, respectively. Using a direct ELISA, dose-dependent levels of DA were detected in each sample matrix tested. RESULTS: An average of 6.6 and 14 ng DA/g brain tissue was found in GD13 and GD20 prenatal rats, respectively. Brain concentrations of DA in the GD13 prenates were identical to amniotic fluid levels, consistent with no restriction for DA to enter the GD13 prenatal brain. At GD20 the prenatal brain contained half the concentration of DA in the amniotic fluid, and was approximately half that found in the brain of the dams. After 1 hr, fetal brain and amniotic fluid contained between 1 and 5% of DA found in the maternal circulation. The amniotic fluid levels of DA in this study were also within the same range measured in stranded California sea lions that showed reproductive failure. CONCLUSIONS: DA crosses the placenta, enters brain tissue of prenates, and accumulates in the amniotic fluid. Amniotic fluid appears to be a useful fluid to monitor DA exposure.

Optimization of blood collection card method/enzyme-linked immunoassay for monitoring exposure of bottlenose dolphin to brevetoxin-producing red tides.

Blood collection cards have been successfully used as a tool to monitor brevetoxin (PbTx) exposure in several species, including fish, mice, and rats. Previous methanolic methods used for extracting brevetoxin from blood collection cards have shown dolphin blood to have matrix difficulties in several biological assays. To better biomonitor protected marine mammal species in the Florida area, which is historically prone to unusual mortality events caused by brevetoxin exposure, we have modified the previous extraction method to consistently recover brevetoxin with a known efficiency from dolphin blood collection card samples with minimal matrix interference. A combination of phosphate-buffered saline (PBS) with 6% MeOH and 100% acetonitrile was used to elute blood from the cellulose card and precipitate proteins, respectively. Analysis was performed using a newly developed direct enzyme-linked immunoassay (ELISA), which yields a sample limit of quantification of 1 ng PbTx-3 equiv/mL. This extraction method allowed for linear recovery of PbTx-3 spiked into dolphin blood (1-30 ng/mL) with a consistent recovery rate of 58% and has subsequently been used to monitor brevetoxins in dolphins, as well as sea turtles and manatees, in regions endemic to red tides. In addition, two known metabolites of PbTx-2 were isolated and also found to be detectable using the ELISA. The cysteine conjugate (m/z 1018) and cysteine sulfoxide conjugate (m/z 1034) were found to have linear recoveries of 87% and 66%, respectively. In summary, this method of extracting brevetoxins and their metabolites from blood collection cards, in conjunction with the ELISA detection method, is a simple and reliable way to biomonitor physiologically relevant toxin levels in protected marine animals.

Domoic acid transfer to milk: evaluation of a potential route of neonatal exposure.

Domoic acid (DA), produced by the diatom genus Pseudo-nitzschia, is a glutamate analog and a neurotoxin in humans. During diatom blooms, DA can contaminate filter-feeding organisms, such as shellfish, and can be transferred by ingestion to higher trophic levels. Several intoxication events involving both humans and various marine mammals have been attributed to DA. Affected organisms show neurological symptoms such as seizures, ataxia, headweaving, and stereotypic scratching, as well as prolonged deficits in memory and learning. Neonatal animals have been shown to be substantially more sensitive to DA than adults. However, it has not been demonstrated whether DA can be transferred to nursing young from DA-exposed mothers. This study demonstrates transfer of DA from spiked milk (0.3 and 1.0 mg/kg) to the plasma of nursing neonatal rats and an overall longer DA retention in milk than in plasma after 8 hr in exposed dams. DA was detectable in milk up to 24 hr after exposure (1.0 mg/kg) of the mothers, although the amount of DA transferred to milk after exposure was not sufficient to cause acute symptoms in neonates.

Ultrasensitive detection of domoic acid in mouse blood by competitive ELISA using blood collection cards.

Domoic acid (DA), an analog of the excitatory amino acid glutamate, is produced by the diatom genus Pseudo-nitzschia and acts as a neurotoxin in humans. During diatom blooms, DA can contaminate shellfish, as well as other filter feeding organisms, and can be transferred by ingestion to higher trophic levels, including marine mammals and humans. The prevalence of this algal toxin and its effects on protected species makes measurement of domoic acid in living animals a necessary biomonitoring tool for the near future. Blood collection cards have already been used for the sampling, extraction and detection of brevetoxin in blood from exposed laboratory animals and, more recently, marine mammals. However, a difficulty unique to measuring DA in blood is the rapid rate (>95% in 2h) at which it is cleared from blood. To meet this challenge, a direct competitive ELISA (cELISA), a method of detection with extremely high sensitivity and specificity, was used to analyze the blood of DA-exposed mice after extraction from the blood collection cards. More than 99% of DA was cleared from blood within 4h post dosage; however, domoic acid was still quantifiable (>0.7ngml(-1)) at 4h from blood spot extracts and still detectable at 24h when compared to control blood spots. By using this highly sensitive assay in conjunction with the use of blood spot cards for easy blood sample extraction, this method could be a very effective means of biomonitoring domoic acid in marine mammals in the field, as well as human populations.