Abstract

Domoic acid is a water soluble tricarboxylic amino acid that acts as an analog of the neurotransmitter glutamate and is a potent glutamate receptor agonist. It is produced by several species of diatom, especially Pseudo-nitzschia australis, which are becoming more common in coastal waters world-wide. Domoic acid activation of the kainate subtype of glutamate receptor results in neuronal cell death in areas of the brain where glutaminergic pathways are heavily concentrated. The CA1 and CA3 regions of the hippocampus, areas responsible for spatial learning and memory processing, are particularly susceptible to domoic acid toxicity, and display extensive lesions in experimentally exposed animals. In California sea lions (Zalophus californianus), natural exposure to domoic acid through ingestion of prey containing domoic acid has resulted in severe neurological signs associated with hippocampal necrosis, and large scale mortality events along the California coast. Cardiomyopathy has also been reported in sea lions following domoic acid exposure, likely due to activation of cardiac kainate receptors. Recently in vitro studies have shown alteration of sea lion lymphocyte function by domoic acid, but it is unclear whether or not this effect is mediated by direct glutamate receptor activation. Blood samples from California sea lions stranding during domoic acid associated mortality events have revealed a high prevalence of circulating eosinophilia, but its significance is unclear.

To investigate the pathogenesis of this eosinophilia and the extent of its association with exposure to domoic acid, hematology, parasite burden and adrenal function were investigated in three groups of sea lions. Six sea lions with clinical signs of acute domoic acid toxicosis^2,4 (seizures, ataxia, hippocampal necrosis, domoic acid in urine), 14 sea lions with clinical signs of long term effects of previous domoic acid exposure² (intermittent seizures, hippocampal atrophy) and 16 sea lions without signs of domoic acid exposure but stranding with unrelated clinical problems (trauma, carcinoma, pneumonia) were included in the study. Each animal was examined clinically; blood, urine and fecal samples were collected for hematology, serum chemistry, circulating cortisol and adrenocorticotrophic hormone (ACTH) levels, domoic acid measurements, and evaluation of worm burden; brain morphology was evaluated by magnetic resonance imaging; and if the animal was euthanized due to poor prognosis, brain and tissues were examined histologically.

Sea lions dying following domoic acid exposure had significantly higher eosinophil counts (mean 929 ± 284/μl in animals dying acutely, 721 ± 188/μl in animals dying chronically) than unaffected animals (320 ± 123/μl). Variability in eosinophil count was high, as five animals unaffected by domoic acid toxicity had eosinophil counts of zero, while only one with acute clinical signs and one with chronic clinical signs had eosinophil counts of zero. Serum cortisol levels were lower in animals following domoic acid exposure (7.2 ± 1.7 μg/dl in acutely affected animals, 10.1 ± 1.3 μg/dl in chronically affected animals) than in unexposed animals (37.6 ± 3.8 μg/dl), as were plasma ACTH levels (53.2 ± 16.5 pg/ml in acutely exposed animals, 50.2 ± 9.4 pg/ml in chronically affected animals and 109.5 ± 26.3 pg/ml in unaffected animals).

Adrenocorticotrophic hormone (2 IU/kg, I.M.) in a long-acting gel preparation (synthesized by Hadfield's Pharmacy, Edmonds WA) was administered intramuscularly to two adult female California sea lions with clinical signs consistent with domoic acid exposure, and fecal samples were collected opportunistically without restraint and evaluated for corticosterone levels using a radioimmunoassay using ¹²⁵I (MP Biomedicals, Costa Mesa, CA). No significant change in fecal corticosterone levels over 3 days were observed in these two animals, whereas ACTH challenge studies in other California sea lions have demonstrated peaks in corticosterone at 42-72 hours post injection (Petrauskas pers. com.).

These results indicate that adrenal function of California sea lions appears to be compromised following exposure to domoic acid. Low serum cortisol levels can explain circulatory eosinophilia independent of parasite burden, as eosinophil counts in humans have been shown to be negatively correlated with serum cortisol. In fact, early assessments of adrenal function were based on eosinophil count.^1,5 Interestingly, serum cortisol is often elevated rather than depressed in humans with temporal lobe epilepsy, but the mechanism for this effect is unclear.³ Future studies will continue to explore the adrenal function in sea lions exposed to domoic acid, as well as investigating the distribution of glutamate receptors in the endocrine system.

Acknowledgements

This work was supported by a grant from the NOAA Prescott program. We thank Lisa Petrauskas for fecal corticosterone assays, and the volunteers of The Marine Mammal Center for their hard work in caring for these animals.

References

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2.  Goldstein T, Mazet JAK, Zabka TS, Langlois G, Colegrove KM, Silver M, Bargu Ates S, Van Dolah F, Leighfield T, Conrad PA, Barakos J, Williams DC, Dennison S, Haulena MA, Gulland FMD 2008. Novel symptomatology and changing epidemiology of domoic acid toxicosis in California sea lions (Zalophus californianus): an increasing risk to marine mammal health. Proc Roy Soc B 275:267-276.

3.  Gallagher BB, Murvin A, Flanigin HF, King DW, Luney D. 1984. Pituitary and adrenal function in epileptic patients. Epilepsia 2S(6): 683-689

4.  Gulland FMD, Haulena M, Fauquier D, Langlois G, Lander ME, Zabka T, Duerr R 2002. Domoic acid toxicity in Californian sea lions (Zalophus californianus): Clinical signs, treatment and survival. Vet Rec 150:475-480.

5.  Thorn GW, Forsham PH, Prunty FTG, Hillas AG 1948. A test for cortical insufficiency. J Am Vet Med Assoc 137:1005-1009.