Domoic acid is a neurotoxic metabolite of widely occurring algal blooms that has caused multiple marine animal stranding events. Exposure to high doses of domoic acid, a glutamate agonist, may lead to persistent medial temporal seizures and damage to the hippocampus. California sea lions (Zalophus californianus) are among the most visible and frequent mammalian victims of domoic acid poisoning, but rapid, reliable diagnosis in a clinical setting has proved difficult owing to the fast clearance of the toxin from the blood stream. Here, we show that the behavioural orienting responses of stranded sea lions diagnosed with domoic acid toxicosis habituate more slowly to a series of non-aversive auditory stimuli than do those of sea lions with no apparent neurological deficits. A signal detection analysis based on these habituation measures was able to correctly identify 50 per cent of subjects with domoic acid toxicosis while correctly rejecting approximately 93 per cent of controls, suggesting potential diagnostic merit

Colleen Reichmuth

Frances Gulland

Peter Cook

CiteSeerX

Animal behaviour
Rapid behavioural
diagnosis of domoic acid
toxicosis in California
sea lions
Peter Cook1,*, Colleen Reichmuth2
and Frances Gulland3
1Department of Cognitive Psychology, and 2Long Marine Laboratory,
Institute of Marine Sciences, University of California Santa Cruz,
CA 95060, USA
3The Marine Mammal Center, Sausalito, CA 94965, USA
*Author for correspondence (pcook@ucsc.edu).
Domoic acid is a neurotoxic metabolite of widely
occurring algal blooms that has caused multiple
marine animal stranding events. Exposure to
high doses of domoic acid, a glutamate agonist,
may lead to persistent medial temporal seizures
and damage to the hippocampus. California sea
lions (Zalophus californianus) are among the
most visible and frequent mammalian victims
of domoic acid poisoning, but rapid, reliable
diagnosis in a clinical setting has proved difficult
owing to the fast clearance of the toxin from the
blood stream. Here, we show that the behavioural
orienting responses of stranded sea lions diag-
nosed with domoic acid toxicosis habituate
more slowly to a series of non-aversive auditory
stimuli than do those of sea lions with no appar-
ent neurological deficits. A signal detection
analysis based on these habituation measures
was able to correctly identify 50 per cent of sub-
jects with domoic acid toxicosis while correctly
rejecting approximately 93 per cent of controls,
suggesting potential diagnostic merit.
Keywords: behavioural diagnostics; habituation;
hippocampus; domoic acid; marine mammals;
sea lions
1. INTRODUCTION
Over the last decade, marine mammal stranding events
coincident with large blooms of Pseudonitschzia austra-
lis have become increasingly common [1]. The factors
producing this increase are complicated, but probably
include interactions between marine mammal feeding
and migratory patterns, and location and timing of
blooms [2]. Some types of Pseudonitschzia diatoms pro-
duce domoic acid, a glutamate agonist with high affinity
for AMPA and kainate receptors [3]. Domoic acid is
cleared from the body rapidly [4], but persistent excito-
toxic effects frequently result in neuronal degradation,
particularly in the hippocampus and surrounding
medial temporal region [5]. Such neuronal necrosis is
particularly acute in the dentate gyrus and hippocampal
sectors CA4, CA3 and CA1 [6,7].
California sea lions have been particularly visible
victims of domoic acid exposure and toxicosis. In
magnetic resonance images (MRIs) of the brain of
42 sea lions diagnosed with chronic domoic acid toxi-
cosis at The Marine Mammal Center in Sausalito, CA,
USA, 41 showed detectable hippocampal atrophy, ran-
ging from mild to severe [1]. Seventy of 89 animals
with chronic domoic acid toxicosis that died during
the same period exhibited gross hippocampal lesions
at necropsy, most commonly in sector CA3 and the
dentate gyrus.
Diagnosing domoic acid toxicosis in a clinical set-
ting is generally a haphazard or time-intensive and
expensive endeavour [8]. Direct diagnosis from blood
sampling is rare as domoic acid is cleared from the
blood stream within 48 h [9], and animals are often
not accessible for treatment until days after exposure
[1]. At The Marine Mammal Center, live animals are
typically suspected of domoic acid poisoning on the
basis of epidemiology and an initial clinical neurologi-
cal examination—greater reliability of diagnosis relies
on post hoc assessment involving laboratory estima-
tion of domoic acid content of urine or faeces, and
analysis of the brain, either by post-mortem histology
or MRI. Both electroencephalography (EEG), which
identifies patterns of seizures, and MRI, which can
identify significant neuronal necrosis and atrophy in
the hippocampus, are effective but relatively slow and
expensive diagnostic tools, and require sedation.
Many cases of domoic acid toxicosis are only accu-
rately diagnosed during post-mortem examination by
histological examination of the hippocampus [6].
Diagnosis determines the course of treatment and
prognosis, and factors into veterinary decisions to
release or euthanize stranded sea lions. Therefore,
improved methods for in vivo diagnosis are needed.
This first attempt at devising a simple and empiri-
cally grounded behavioural diagnostic assay of
domoic acid toxicosis depends on the habituation of
an orienting response to non-aversive auditory stimuli.
As hippocampal necrosis is a common consequence of
domoic acid toxicosis in sea lions, and hippocampal
damage has been shown to slow habituation rates of
unrestricted exploratory behaviour in a range of species
[10–12], a metric based on habituation holds diagnos-
tic promise. Further, the habituation of an orienting
response can be measured through observation, requir-
ing no invasive or aversive procedures [13]. Here, we
present the results of a behavioural assessment
designed to be sensitive to hippocampal damage in
order to augment diagnosis of domoic acid toxicosis
in sea lions.
2. MATERIAL AND METHODS
Forty-two sea lions undergoing rehabilitation at The Marine
Mammal Center were sampled in this study (see the electronic
supplementary material, subject table). Effort was made to test all
available admitted sea lions, regardless of diagnosis, during the
study period. After testing was complete, subjects were assessed
using veterinary clinical criteria wholly independent of performance
in testing. Twelve sea lions were diagnosed with domoic acid toxico-
sis and 27 sea lions, forthwith referred to as ‘controls’, were
evaluated as having no signs of domoic acid toxicosis or other neuro-
logical abnormalities. A positive diagnosis of domoic acid toxicosis
was based either on clinical signs of seizures and ataxia that resolved
following diuresis and sedation (as described in [6]) and presence of
domoic acid in urine or faeces; or on detection of an abnormal hip-
pocampus through MRI or post-mortem histology. Three sea lions
had indeterminate diagnoses and were not included in the final
sample.
Electronic supplementary material is available at http://dx.doi.org/
10.1098/rsbl.2011.0127 or via http://rsbl.royalsocietypublishing.org.
Biol. Lett.
doi:10.1098/rsbl.2011.0127
Published online
Received 1 February 2011
Accepted 16 February 2011 This journal is q 2011 The Royal Society
 on March 15, 2011rsbl.royalsocietypublishing.orgDownloaded from 
The behavioural assessments were conducted in a quiet pen. Fol-
lowing acclimation, each sea lion was exposed to a series of auditory
stimuli in four sequential test phases. The testing sequence was
designed to examine initial habituation of orienting responses to
novel auditory stimuli, and to probe recovery of response, or dishabi-
tuation, following manipulations of spatial presentation, recovery
interval and stimulus type. Each test phase comprised successive pre-
sentations of one of two sounds from one of two diametrically
opposite locations (see the electronic supplementary materials for
further details on experimental manipulations).
During testing, an experimenter, blind to subject diagnosis,
observed subject behaviour and coded responses in real time from
closed circuit video. An orienting response emitted following stimu-
lus onset, and within 0.5 s of stimulus offset, was considered a
positive response. Orienting was defined as a noticeable change in
the angle of the subject’s head towards the source of the stimulus
in the vertical or horizontal plane. During each of the four testing
phases, the auditory stimulus was presented on a fixed, semi-
random schedule at intervals of 5–15 s until the subject habituated,
at which point the next phase began. ‘Habituation’ was defined as no
observable orientation to three consecutive stimuli. The exper-
imenter’s real-time assessment of habituation was used during
testing and for subsequent analysis. These scores were later validated
by two independent observers who viewed the videotaped recordings
of each session.
Exposures to habituation were compared between sea lions with
domoic acid toxicosis and controls for each of the four test phases
using t-tests with Bonferroni corrections for repeated measures.
Test phases showing a significant difference between these groups
were then further subjected to signal detection analysis employing
receiver operating characteristic (ROC) curves. ROC curves assess
a metric’s likelihood of producing a correct positive diagnosis relative
to the likelihood of a false positive diagnosis over a range of diagnos-
tic sensitivity thresholds. Thresholds used here were the number of
exposures prior to habituation in a particular test phase.
3. RESULTS
Sea lions with domoic acid toxicosis took significantly
more exposures to habituate in the first test phase
than did controls (figure 1)—there were no significant
differences observed in test phases 2–4. Agreement
between the experimenter’s initial coding of orienting
behaviour across all subjects and exposures and that
of the post hoc observers was 85 and 86 per cent.
A ROC curve was computed using independent
diagnosis of domoic acid toxicosis and the number of
exposures to habituation in phase 1 (figure 2). The
area under the curve was 0.82, suggesting a good
diagnostic metric.
4. DISCUSSION
The notable tendency of sea lions with domoic acid
toxicosis to habituate more slowly to a non-aversive
auditory stimulus may be explained by the presence
of hippocampal damage in these subjects. There were
no significant differences in responsiveness between
subject groups in phases 2–4, suggesting that the
spatial, delay and stimulus manipulations did not
have differential effects on animals with domoic acid
toxicosis. More generally, this result indicates that
these dishabituation measures were relatively insensi-
tive to confirmed or presumed hippocampal damage.
Quantitative behavioural diagnostics are rarely used
in veterinary clinical settings, but in this case, ROC
analysis of exposures to habituation in the first test
phase indicates that habituation is a promising measure
to augment current diagnostic approaches to domoic
acid toxicosis. ROC analysis produces a ratio of correct
positive diagnoses to false positive diagnoses across a
range of thresholds (see the electronic supplementary
material, ROC criteria table). In the case of domoic
acid toxicosis in sea lions, false diagnosis of domoic
acid toxicosis could lead to an otherwise healthy
sea lion’s being euthanized (as recommended in [14]),
so ultimately, a conservative diagnostic threshold of
greater than 22 exposures prior to habituation was
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20
30
40
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Figure 1. Responsiveness across auditory phases.Meannumber
of exposures prior to habituation for subjects diagnosed with
domoic acid toxicosis and controls in four test phases: initial
exposure, exposure following a spatial shift, exposure following
a delay and exposure following a stimulus shift. Error bars rep-
resent standard deviation. There was a significant difference
(asterisk) between exposures to habituation for sea lions with
and without domoic acid toxicosis in the first (p, 0.001) but
not the second, third or fourth test phases (p. 0.05, t-tests
with Bonferroni correction for repeated sampling). White bars,
control; black bars, domoic acid.
20 40 60 80 1000
20
40
60
80
100
% false positive diagnoses
%
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Figure 2. ROCcurve based on exposures prior to habituation in
test phase 1.ThisROCcurve represents the diagnostic effective-
ness of a metric based on exposures prior to habituation in the
first test phase. The likelihood of producing a correct positive
diagnosis (represented on the y-axis) versus the likelihood of a
false positive diagnosis (represented on the x-axis) was com-
puted across a range of sensitivity thresholds. These thresholds
were defined as the number of exposures prior to habituation
above which an animal was considered to have domoic acid tox-
icosis. For the ROC curve, diagnosis was considered correct if it
matched the independent veterinary assessment, and incorrect if
it did not. The area under the curve was 0.82 and p, 0.002.
2 P. Cook et al. Rapid behavioural diagnosis in sea lions
Biol. Lett.
 on March 15, 2011rsbl.royalsocietypublishing.orgDownloaded from 
selected for this study. Using this threshold, the habitu-
ation measure correctly identified 50 per cent of sea
lions with domoic acid toxicosis while falsely diagnosing
only 7 per cent of control subjects. This auditory
response test can serve as a rapid, inexpensive and logis-
tically easy diagnostic test for hippocampal damage
available to most practitioners in the absence of
advanced and expensive clinical diagnostics such as
MRI or EEG. This represents a novel and applicable
behavioural approach to diagnosis of a neurological dis-
order in a veterinary setting.
Further refinement of the procedure is ongoing in
concert with a study of sea lions with domoic acid
toxicosis in which all subjects are undergoing MR
brain imaging. This may improve an already effective
diagnostic measure, and will indicate whether the
behavioural assay discussed here is sensitive to hippo-
campal damage as suspected or to other sequelae of
domoic acid toxicosis.
This research was conducted under authorization granted to
the Marine Mammal Health and Stranding Response
Programme by NMFS under scientific research permit
932-1489-10. This research has been approved by the
Institutional Animal Care and Use Committees at both
UCSC and TMMC.
We thank Ronald Schusterman and Kristy Lindemann for
assistance in experimental design; Nicola Pussini and Lauren
DeMaio for logistical support at TMMC; TMMC
veterinarians and staff for diagnostic work; William Hughes
and Austin Paul for post hoc assessment; and Andrew Rouse
for assistance with the manuscript preparation. This work was
supported by a Packard Endowment for Ocean Sciences and
Technology award, and P.C. was supported by a National
Science Foundation Graduate Research Fellowship.
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Rapid behavioural diagnosis of domoic acid toxicosis in California sea lions

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Rapid behavioural diagnosis of domoic acid toxicosis in California sea lions

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