Domoic acid toxicity in Californian sea lions

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The detection of saxitoxin in dead humpback whales. (Megaptera novaeangliae) offNew England resulted in the first confirmed case of a biotoxin affecting a ...

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Domoic acid toxicity in Californian sea lions (Zalophus californianus): clinical signs, treatment and survival F. M. D. GULLAND, M. HAULENA, D. FAUQUIER, G. LANGLOIS, M. E. LANDER, T. ZABKA, R. DUERR Eighty-one Californian sea lions (Zalophus californianus) with signs of domoic acid toxicity stranded along the coast of California in 1998 when there were blooms of the domoic acid-producing alga Pseudonitzschia australis off-shore. In 2000, a further 184 sea lions stranded with similar clinical signs, but the strandings occurred both during detectable algal blooms and after the blooms had subsided. The clinical signs in these 265 Californian sea lions included seizures, ataxia, head weaving, decreased responsiveness to stimuli and scratching behaviour. Affected animals had high haematocrits, and eosinophil counts, and high activities of serum creatine kinase. They were treated supportively by using fluid therapy, diazepam, lorazepam and phenobarbitone. Fifty-five of the 81 sea lions (68 per cent) affected in 1998 and 81 of the 184 (44 per cent) affected in 2000 died despite the treatment. Three of the 23 sea lions which survived in 1998 were tracked with satellite and radiotransmitters; they travelled as far south as San Miguel Island, California, and survived for at least three months. Eleven of the 129 animals which were released stranded within four months of being released.

Veterinary Record (2002) 150, 475-480 E M. D. Gulland, VetMB, PhD, M. Haulena, DVM, MSc, D. Fauquier, DVM, M. E. Lander, MSc, T. Zabka, DVM, R. Duerr, The Marine Mammal Center, 1065 Fort Cronkhite, Sausalito CA 94965, USA G. Langlois, PhD, Department of Public Health, University of California at Berkeley, CA 94720, USA

TOXIC algal blooms are well recognised as causes of human and animal morbidity and mortality (Baden and others 1995). Their role in the mortality of marine mammals is often uncertain owing to the logistical difficulties associated with investigating the deaths of marine mammals, and the lack of data on the toxic thresholds of marine toxins in these species. The detection of saxitoxin in dead humpback whales (Megaptera novaeangliae) off New England resulted in the first confirmed case of a biotoxin affecting a marine mammal (Geraci and others 1989). Brevetoxicosis was later identified as the cause of an epizootic that affected manatees (Trichechus manatus latirostris) off Florida in 1996 (Bossart and others 1998) and it had probably been the cause of earlier epizootics in 1946 and 1982 (Gunter and others 1948, O'Shea and others 1991). Other cases of biotoxin-associated mortality are less clear. Brevetoxin was identified in tissues of bottlenose dolphins (Tursiops truncatus) that died off the eastern USA in 1987 and 1988 (Geraci 1989), and saxitoxin was found in the tissues of Mediterranean monk seals (Monachus monachus) that died in 1998 (Costas and Lopez-Rodas 1998). The role of biotoxins in these two die-offs has been disputed (Harwood 1998, Hernandez and others 1998). In all these cases, live affected animals were rarely seen, and the clinical signs and treatment of affected animals have not been recorded. In 1998, domoic acid toxicity was determined to be the cause of death of 48 Californian sea lions (Zalophus californianus) that stranded along the coast of California during a bloom of the diatom Pseudonitzschia australis (Scholin and others 2000). Domoic acid is a marine toxin produced by a number of marine algae (Iverson and Truelove 1994, Bates 1998). It was identified as a cause of toxicosis in people who had eaten contaminated mussels off the east coast of Canada (Wright and others 1989, Perl and others 1990). In 1991, it was first reported as a threat to the health of wildlife in the Pacific when it was associated with the deaths of pelicans (Pelicanus occidentalis) (Work and others 1993). Since then, blooms of domoic acid-producing algae have been observed regularly off the central Californian coast (Walz and others 1994) and they were first shown to affect marine mammals in 1998 (Scholin and others 2000). Despite these reports of the deaths of marine mammals due to harmful algal blooms, there is no information either about the clinical signs and methods for treating the affected animals, or about the success of any treatment. Harmful algal

The Veterinary Record, April 13, 2002

blooms can rapidly affect large numbers of animals, can cause severe clinical signs and death, and can affect threatened and endangered species. To develop plans for responding to such blooms, data on previous attempts at treatment and their success are needed. This paper describes the clinical signs, treatment and survival of Californian sea lions poisoned by domoic acid associated with two algal blooms off the central Californian coast in 1998 and 2000. MATERIALS AND METHODS

Californian sea lions that stranded along the Californian coast between 37°42'N, 123°05'W and 35°59'N, 121°30'W were transported to the Marine Mammal Center, Sausalito, for rehabilitation. Upon arrival the animals were examined, their sex determined and their age class estimated from their body length, bodyweight and degree of tooth and sagittal crest development (Mate 1978). As depression and coma were common clinical signs, an objective method for assessing the clinical responsiveness of the sea lions was developed and the results were recorded on a standard form (Fig 1) which was completed after the examination of each animal and a score was assigned to it. This score was determined for each animal on admission, then every two to three days and then at longer intervals as the animals recovered. Blood samples were collected from the caudal gluteal vein with 1-5 inch 21 G needles directly into vacutainers containing either ethylenediamine tetra-acetic acid (EDTA), or serum separation gel and clot activator (Vacutainer Systems; Becton Dickinson) (Bossart and others 2001). Complete blood counts were obtained on samples in EDTA with a haemocytometer (Heska), and serum biochemical measurements were made on a Vet Test 8008 (Idexx Laboratories). The variables determined included total and differential white cell counts, red cell count, haemoglobin, haematocrit, platelet count and serum levels of sodium, potassium, chloride, glucose, blood urea nitrogen, creatinine, total protein, albumin, globulin, uric acid, calcium, phosphorus, total and indirect bilirubin, gamma-glutamyl transferase, alkaline phosphatase, alanine and aspartate aminotransferase, lactate dehydrogenase, iron, cholesterol, triglycerides and creatine kinase. Creatine kinase isoenzymes were determined by electrophoresis. Haematocrit, eosinophil and creatine kinase values from affected sea


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lions were compared with values from samples collected from apparently healthy Californian sea lions which were being rehabilitated after traumatic injuries, by using t tests with the statistical software Minitab. Serum samples from 34 animals showing clinical signs were analysed by virus microneutralisation for the presence of antibodies to phocine distemper virus (PDV) at the United States Department of Agriculture laboratory on Plum Island (Duignan and others 1994). Urine samples were collected from free-flowing urine when animals urinated while being restrained for clinical examinations. The samples were stored at -80°C before being sent to the National Oceans Service laboratory in Charleston, South Carolina, and the National Marine Fisheries Service laboratory in Seattle, Washington, where they were tested for domoic acid by receptor-binding assay, with selected samples being analysed by liquid chromatography mass spectrometry to confirm the identity of the domoic acid (Scholin and others 2000). Faecal samples were collected from the pen floor directly after they were voided and tested for domoic acid by high performance liquid chromatography (HPLC) (Lefebvre and others 1999). Two animals were anaesthetised with a medetomidine/ zolazepam/tiletamine mixture and isoflurane and examined by magnetic resonance imaging (Mm) (Haulena and Gulland 2001). Sea lions that died were examined postmortem and samples of all their organs were fixed in 10 per cent neutralbuffered formalin for histopathological examination. All the surviving sea lions were tagged in the fore flipper with plastic cattle tags (Dalton) before being released. To monitor the movements and survival of sea lions which had suffered seizures induced by domoic acid, three adult females (A, B and C) were tagged with satellite transmitters during 1998; C had seizures for 48 hours and then recovered after treatment, A had repeated seizures for up to a week before recovering, and B had seizures for up to one month before recovering. The three sea lions were sedated with 0-2 mg/kg midazolam (Versed) injected intramuscularly with a 1-5 inch 21 G needle and 6 cm3 syringe into the gluteal muscles (mixed with 0-2 mg/kg atropine), then masked with isoflurane until relaxed, but with intact gag reflexes. Satellite-linked transmitters (ST-10 model; Telonics) were glued to the dorsum between the scapulae with five-minute epoxy (Devcon). Because the tags were glued to the sea lions' pelage, the animals were not tagged until November 4, after the annual moult. They were released on November 6 at Point Lobos State Reserve, Carmel, California, and their daily positions (location classes 0 to 3) were obtained by using Service Argos, a satellite-based computerised tracking system, and plotted by using the computer software ArcView 3-0 (ESRI) and Animal Movement, an ArcView extension (Hooge and Eichenlaub 1997), after screening the data for erroneous positions (Merrick and Loughlin 1997).




Acc No:


Total Score:


1. 2. 3. 4.

Mentation 1. Normal 2. Confusion/Disorientation 3. Obtunded (responsive) 4. Coma (unresponsive)

Abnormal body movements (each assigned 1 point) Ocular: Nystagmus: vertical/horizontal/postural/fixating/continuous Strabismus: note position Rapid blinking Grimacing Excessive chewing motion Scratching Muscle fasciculations: note muscle/body part affected Myoclonus (tremor-like activity) Myotonus (onger duration muscle contractions) Chorea (rapid, jerky, purposeful movements) Athetoid (slow, writhing, purposeless movements)

Responsiveness To audible or visual appzvach 1. Charges person aggressively 2. Moves away at speed 3. Approaches but non-threatening (curiosity/confusion) 4. Moves away hesitantly 5. Lifts head 6. Moves eyes to observe 7. Non-responsive 8. Seizures: note type of movements above To touching/pinching lumbar azra Too aggressive for manipulation Moves away rapidly Approaches but non-threatening (curiosity/confusion) Moves away slowly Attempts to move but unable Lifts head Non-responsive Seizures: note type of movements above

1. 2. 3. 4. 5. 6. 7. 8.

Locomotion 1. Able to move rapidly using 4 flippers 2. Able to move slowly/reluctantlyusing 4 flippers 3. Moves without use of hind flippers/Scoots on ventrum without using fore flippers 4. Ataxic/ Uncoordinated movements 5. Attempts to move but can't rise 6. Unable to use fore and hind flippers on same side (hemiplegia) 7.

RESULTS Between May 15 and June 15, 1998, 70 Californian sea lions showing signs of domoic acid toxicity stranded live along the central Californian coast and were transported to the Marine Mammal Center for rehabilitation; between July 1 and October 8, 1998, an additional 11 Californian sea lions stranded, showing similar clinical signs, and between June 23 and December 1, 2000, 184 sea lions showing similar clinical signs stranded (Fig 2). A diagnosis of domoic acid intoxication was made for the animals affected in these three stranding events. In 1998, the diagnosis was based on the characteristic clinical signs, the presence of domoic acid in their urine and faeces, the histopathological changes in the animals that died, the presence of a bloom of P australis producing domoic acid 476

Sitting up on fore flippers Recumbent: sternal, dorsal, lateral: R/L Body twisted: R/L Opisthotonos (head pointing upward, limbs and body in rigid extension)


8. Immobile FIG 1: Clinical assessment sheet for evaluating Californian sea lions with neurological signs. R Right, L Left in the waters the sea lions were swimming in, and the presence of domoic acid in prey they were eating (Lefebvre and others 1999, Scholin and others 2000). In 1998, the strandings coincided with periods of intense Pseudonitzschia species blooms off the central Californian coast, but in 2000, the strandings did not always coincide with blooms, and the presence of blooms did not always result in stranded sea lions showing signs of domoic acid toxicity (Fig 2). Of the 81 sea lions affected in 1998, 64 were adult females, five were juvenile females, 11 were juvenile males and one was

The Veterinary Record, April 13, 2002

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FIG 2: Relationship between the numbers of stranded Californian sea lions admitted tI 0 the Marine Mammal Center per week with clinical signs of domoic acid toxicity and th e abundance of Pseudonitzschia australis (a) in 1998 at Santa Cruz Pier, California, and (b) in 2000 at Port San Luis, Diablo Cove, Califomia. P australis abundance: 1 Rare, 2 Pres ent, 3 Common, 4 Abundant

a yearling female; no adult males were affected. In 2000, 14 7 of the 184 animals were adult females, nine were adult males , 10 were juvenile females, 15 were juvenile males and three uvere yearling females. They were all in good nutritional conditiion. The characteristic clinical signs were ataxia, head weav ing, muscle tremors, tetanic convulsions and coma. In the aninnals that died, seizures became increasingly frequent, often F)rogressing to status epilepticus before death. If the animals uvere repeatedly in status epilepticus and showed no improvemient in clinical signs over five days, they were euthanased. The frequency of seizures varied from one to 30 in 24 hours. In the animals that survived, the frequency of seizures graduially decreased over a period of a month. The seizures were usuially

The Veterinary Record, April 13, 2002

bilateral, but two animals repeatedly had unilateral seizures. These consisted of muscle spasms on one side of the body, the lips being retracted to the affected side, and spasm of the eyelids on the affected side only. Between seizures, the sea lions became lethargic to varying degrees and occasionally developed a characteristic rubbing behaviour, in which they would rub the back of their necks and backs by rolling repeatedly on the floor of the pen. Animals that did not have seizures were depressed to varying degrees, extreme cases becoming unresponsive to stimuli. The degree of unresponsiveness did not change in some animals that were eventually euthanased, whereas other animals gradually improved clinically. The treatment of the animals modified their clinical signs and condition scores. Scores were not available for all the animals before medication started, but those with a score under 15 survived the toxicosis, whereas 13 of the 18 (72 per cent) with an admission score over 25 died, and 19 of the 46 (41 per cent) ofthose with admission scores between 15 and 24 died. The scores tended to remain over 15 until treatment with phenobarbitone ceased. The mean haematocrits and eosinophil counts were significantly higher in sea lions with clinical signs of domoic acid toxicity than in unaffected clinically normal sea lions bled before they were released. The mean haematocrit in 201 affected animals was 49-5 per cent, compared with 42-8 per cent in 144 clinically normal adult sea lions (P