Case Report

Veterinary Pathology
2016, Vol. 53(5) 1103-1106
Integrating the Forensic Sciences in Wildlife ª The Author(s) 2016
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Case Investigations: A Case Report DOI: 10.1177/0300985816641176
vet.sagepub.com
of Pentobarbital and Phenytoin Toxicosis
in a Bald Eagle (Haliaeetus leucocephalus)

T. C. Viner1, B. C. Hamlin1, P. J. McClure1, and B. C. Yates1

Abstract
The application of medical knowledge to the purpose of law is the foundation of forensic pathology. A forensic postmortem
examination often involves the expertise of multiple scientific disciplines to reconstruct the full story surrounding the death of an
animal. Wildlife poses additional challenges in forensic investigations due to little or no associated history, and the disruptive effects
of decomposition. To illustrate the multidisciplinary nature of wildlife forensic medicine, the authors outline a case of secondary
pentobarbital/phenytoin toxicosis in a bald eagle (Haliaeetus leucocephalus). The eagle was the single fatality in a group of 8 birds that
fed on euthanized domestic cat remains that had been improperly disposed of in a landfill. Cooperation between responding law
enforcement officers, pathologists, and other forensic scientists led to the successful diagnosis and resolution of the case.

Keywords
eagle, forensics, genetics, morphology, pentobarbital, toxicity, wildlife

The practice of diagnostic pathology—the assessments used to report of fatal pentobarbital and phenytoin toxicosis in a bald
discover and delineate the disease or condition that caused an eagle (Haliaeetus leucocephalus) that was examined at the US
animal’s death—is a multimodal exercise. Infrequently is a Fish and Wildlife Service’s National Fish and Wildlife Foren-
definitive cause-of-death judgment based on gross or micro- sics Laboratory (NFWFL). Eight adult and subadult bald eagles
scopic examination alone. Gross findings must be married with were found in and near a municipal landfill site. At the time of
microscopic, toxicologic, microbiologic, and/or clinicopatho- discovery, the eagles exhibited signs of neurologic depression,
logic findings to build the full and complete story about regurgitation, and/or convulsions. Several eagles were
the death of that animal. The case is similar in the practice of depressed to the point of appearing to be dead to onlookers.
forensic pathology. To fully discover the ‘‘who, what, when, Staff from a wildlife rehabilitation program collected 7 of the
where, and how’’ of a veterinary forensic pathology story, mul- 8 eagles; 1 eagle flew away during the capture attempts. The
tiple aspects of the forensic sciences are often utilized. Sciences 7 captured birds received supportive treatment and showed
that may contribute to a full forensic analysis—filling in the improvement over the next several days. Samples of regurgi-
outline provided by a forensic pathology examination—include tated material were frozen for potential analysis.
genetics, morphology, toxicology, and criminalistics. Examination of the landfill site around where the eagles
Domestic animals, by definition, live in close proximity to were found revealed the partially scavenged remains of 2 mam-
humans. Their health and well-being depend on the presence and mals appearing to be domestic cats (Fig. 1). The remains were
assistance of people. Investigation of cases of death due to found uncontained and exposed to the environment.
neglect, therefore, involves suspects and crime scenes not too
far from the victim. In contrast, wild animals are independent,
often avoiding civilization and places that humans frequent.
Because of this separation, linking a suspect to a wildlife victim 1
National Fish and Wildlife Forensics Laboratory, Ashland, OR, USA
can be problematic. Adding to this challenge, and inherent to the
separation between wildlife and humans, is an often extended Supplemental material for this article is available on the Veterinary Pathology
time between death and discovery of the remains. Autolysis and website at http://vet.sagepub.com/supplemental.
the effects of scavenging often obscure or remove changes in the
Corresponding Author:
body that could tell the story of an animal’s death. T. C. Viner, National Fish and Wildlife Forensics Laboratory, 1490 E Main St,
To illustrate the multidisciplinary nature of wildlife forensic Ashland, OR 97520, USA.
pathology and the challenges that may arise, we present a case Email: [email protected]
1104 Veterinary Pathology 53(5)

Figure 1. Domestic cat. Mammalian remains found at the municipal landfill site and morphologically determined to be of domestic cat origin.
Eight eagles were found exhibiting signs of CNS disturbance in proximity to these remains. Figure 2. Adult bald eagle (Haliaeetus leucocephalus).
The bird was found dead near a snowmobile trail 25 miles from a landfill site where euthanized cats were found exposed to the environment.
Ventral recumbency and spread wings indicate that the eagle had been in a relaxed state prior to death. This is in contrast to the flexed death
pose regularly seen in organophosphate or carbamate poisoning cases.

Later the same day, a dead eagle was found on a snowmo- Results of the chemical analyses of the eagle’s postmortem
bile trail located 25 miles from the municipal landfill site ventriculus contents and antemortem regurgitated material
(Fig. 2). The eagle appeared to have regurgitated prior to death. indicated that the barbiturate pentobarbital and the anticonvul-
The body was prone and the wings of the eagle were spread sant phenytoin were present in both samples.
indicating a relaxed state prior to death. Law enforcement per- Forensic genetic analysis was conducted to determine the
sonnel responding to both scenes suspected that the eagle that species of origin of the contents of the regurgitated material .
flew away during capture attempts at the landfill site was the DNA was extracted from a subsample of the regurgitated mate-
same eagle found on the snowmobile trail. The dead eagle and rial and a segment of the mitochondrial 16 S rDNA gene was
regurgitated material were submitted with the remains of the amplified by PCR (Short-16S-F: gAAATTgACCTTCCCgT-
mammal by the responding law enforcement officer to NFWFL gAAgAgg; Short-16S-R: CgCTgTTATCCCTAGggTAACT)
for cause of death determination. and subjected to Sanger sequencing by capillary electrophor-
The eagle underwent a forensic postmortem examination esis. The resulting 212 base pair 16 S rDNA sequence was
which included radiographs, complete skinning of the carcass, compared to the NFWFL mammal reference sequence
and removal of the viscera. The ventriculus contents were col- database.
lected in aluminum foil, officially sealed, and submitted for A 212 base pair segment of the 16 S rDNA locus of mito-
chemical analysis. Per standard forensic analysis guidelines, chondrial DNA was amplified from DNA extracted from the
photographs were taken to document both normal and abnor- regurgitated material. This sequence was compared to those in
mal gross findings. the NFWFL mammal reference sequence database and found to
The eagle weighed 4.5 kg just prior to necropsy and had a be identical to that of domestic cat (Felis catus) at 212/212
good postmortem preservation status. Musculature over the bases. The sequence was compared to and found to be unlike
shoulders and chest was moderately depleted, but fat stores were those of other species in the genus Felis, as well as other North
present in adequate amounts. Ventriculus contents consisted American wild cats including cougar, bobcat, and lynx.
of only 2 ml of dark green, mucoid ingesta. Small amounts of Forensic morphology examination was used to identify
digesta were present in the intestinal tract. Aspirated ingesta was mammalian remains found at the landfill site and considered
present in the tracheal lumen. The lungs, liver, heart, brain, and a probable source of any toxic material. The remains con-
kidneys were unremarkable. There was no gross evidence of sisted of mangled limbs and partial cranium covered in white
trauma, gunshot wounds, electrocution, or systemic disease. fur. Standard morphological protocols were used to determine
Samples for the forensic chemistry examination consisted of the taxonomic identification of these remains. Analysis con-
regurgitated material from an eagle that survived, and the ven- sisted of extracting those anatomical elements that offer diag-
triculus contents from the bald eagle submitted for necropsy. nostic details at the species level, which in this case included
These samples, as well as a control sample, were subjected to dentition and claws.
extraction with methylene chloride, and then a portion of each Dental observations of the mammalian remains revealed
extract was concentrated to dryness. Triphenyl phosphate was lateral grooves on the canine teeth, as well as retractile claws
added as an internal standard and the samples were analyzed on the feet. Both of these features are diagnostic for felids. Size
employing an Agilent 6890 Gas Chromatograph coupled with and fur color completed the morphological determination of
an Agilent 5973 Mass Spectrometer. domestic cat as the species represented by the remains.
Viner et al 1105

Based on the combined forensic analyses, it was determined llama or alpaca. Chemical analysis of liver or stomach contents
that the eagle submitted to the NFWFL for necropsy had died of the affected animals showed both pentobarbital and pheny-
of pentobarbital and phenytoin toxicosis through ingestion of a toin in 11 of the 62 animals; pentobarbital alone was detected in
euthanized cat at a municipal landfill site. The regurgitated 50 animals, and the bobcat had ingested phenobarbital. The
material found with the dead eagle, and the eagle’s ventriculus combination of pentobarbital and phenytoin is present in sev-
contents both contained pentobarbital and phenytoin. Though eral veterinary formulations for domestic animal euthanasia,
the eagle’s body was found 25 miles from the landfill site, the including Beuthanasia-D Special (manufactured by Intervet/
eagle could be placed at the site through genetic analysis of Merck Animal Health) and Euthasol Euthanasia Solution (man-
the eagle regurgitated material and morphological analysis of ufactured by Virbac).
the mammalian remains at the landfill, both of which were Each state within the United States creates and administers
determined to be of domestic cat origin. Histology was deter- laws regarding the disposition of euthanized animals. Disposal
mined to be unnecessary in the determination of the eagle’s practices outlined in the laws are designed to prevent negative
cause of death and was not performed. The 7 eagles that were impacts to wildlife and the environment by making the animals
initially captured and treated fully recovered within 2 months and tissue drug residues unavailable for ingestion or contam-
and were released into the wild. ination of the environment. It is the responsibility of the veter-
The NFWFL does not conduct genetic matching (individual inarian licensed to administer euthanasia solutions to either
identity) analysis of domestic species such as cats, as this anal- dispose of a euthanized animal appropriately, or advise staff
ysis falls outside the mission scope of the laboratory. Genetic at a holding entity, such as an animal shelter, of correct disposal
matching of many domestic species is available at other insti- practices.
tutions.1 In the case presented here, the circumstantial evidence The successful resolution of a forensic pathology case is
of domestic cat tissue and pentobarbital in the dead eagle and rarely an individual effort. As in this case example in which
regurgitated material, taken in conjunction with events and several scientific disciplines added pieces of data to determine
findings at the municipal landfill crime scene was considered the cause of the eagle’s death, reconstructing the events sur-
to be adequate for successful prosecution of the case. rounding the death of an animal often requires the efforts of
Incidents of pentobarbital toxicosis of wildlife have been more than 1 analyst.
rarely documented in the literature.4,5 A retrospective survey As in clinical medicine and diagnostic pathology, a good
of cases at the US Geologic Survey’s National Wildlife Health investigation starts with a good history. Unfortunately, in many
Center in Madison, Wisconsin, showed that 4.3% of bald eagle wildlife cases, a clinical history is unavailable as animals may
poisonings and 2.6% of golden eagle (Aquila chrysaetos) poi- be found dead in the environment. A thorough investigation of
sonings were due to barbiturate intoxication.5 From this study the scene by the responding law enforcement personnel may
population of 2980 bald eagles and 1427 golden eagles that glean information or evidence that can help the investigation.
were submitted for necropsy, 33 bald eagles and 3 golden Communication of these findings, as well as the location of the
eagles succumbed to barbiturate poisoning.5 Langelier reported body (eg, under a power pole or near a baited carcass) can help
an incident involving 29 bald eagles that fed on a euthanized the veterinary pathologist direct his/her analysis. In the case
domestic cow.4 Of this group, 5 were found dead or died within presented here, the law enforcement officer relayed to the
an hour of presentation, and 1 was found 21 km from the cow veterinary pathologist information about the possible link
carcass. As exemplified by Langelier and the bald eagle in this between the eagle found dead at the remote snowmobile trail
case report, the clinical course of pentobarbital toxicosis may and the clinically affected eagles at the landfill site. This infor-
be such that birds can fly or soar great distances from the point mation allowed the veterinary pathologist to refine a list of
source of the toxin. Removal from the scene of the crime elim- possible causes of death and prioritize tests that would support
inates environmental and proximity clues that could assist an or disprove the primary hypothesis.
investigation. Diligent and thorough analysis of these cases is Other challenges that wildlife forensic pathologists fre-
required to provide an accurate diagnosis. quently face are the effects of decomposition and scavenging.
A review of case records dating from 1992 to 2015 at the Wild animals rarely die at the doorstep of a veterinary clinic or
NFWFL revealed 62 animals with suspected or definitive rehabilitation center where their remains can be either appro-
mortality due to secondary barbiturate intoxication in a total priately preserved or expeditiously examined. Wild animals
of 41 separate events (Supplemental Table 1). Animals diag- tend to die in remote or hidden venues, exposed to the factors
nosed with fatal barbiturate intoxication included bald eagles of weather, climate, and opportunistic feeders. These agents
(40), golden eagles (11), 2 red-shouldered hawks (Buteo line- effect changes on the body—a process termed ‘‘taphonomy’’—
atus), 1 Cooper’s hawk (Accipiter cooperii), 1 red-tailed hawk that may alter or obscure evidence that points toward a cause
(Buteo jamaicensis), 1 black-billed magpie (Pica hudsonia), of the animal’s death.
1 common raven (Corvus corax), 1 coot (Fulica americana), Taphonomic processes may render a carcass unidentifiable
1 common crow (Corvus brachyrhynchos), 1 bobcat (Lynx to the untrained eye. Invertebrate and vertebrate scavengers
rufus), and 1 coyote (Canis latrans). Species that were pre- may deflesh bones and scatter identifying characteristics. Even
sumptively euthanized and eaten by the affected animals with highly altered remains, depending on the extent of degra-
included horse, mule, cat, sheep, goat, cow/calf, dog, pig, and dation, certain modalities may be used to (1) identify the
1106 Veterinary Pathology 53(5)

species and (2) determine cause of death. In the case report challenges and lead to the successful resolution of a veterinary
herein, genetic and morphologic examination of heavily sca- forensic pathology investigation.
venged remains revealed domestic cat as the origin of the toxin
that affected the eagles. Muscle remnants, regurgitated mate- Acknowledgements
rial, teeth, and claws were all that were needed for genetic and We wish to thank US Fish and Wildlife Service Special Agent
morphologic species assignment. S. Stoinski for investigation of the pentobarbital case and
Pathologic processes may remain evident even in highly submission of case materials, and Colleen Wilson for pathology
degraded remains, but must be differentiated from changes technical assistance. The findings and conclusions in this article are
induced by aggressive scavengers, such as rodents and those of the authors and do not necessarily represent the views of
canids.2,3 While bullets often cause perforating or excoriating the US Fish and Wildlife Service.
lesions in bone, so too can scavenging activity. Use of an x-ray
florescence analyzer may reveal minute particles of lead that Declaration of Conflicting Interests
are neither grossly visible nor evident on standard radiographs The author(s) declared no potential conflicts of interest with respect
of heavily degraded remains. The presence of lead particles to the research, authorship, and/or publication of this article.
around a fracture strongly supports a diagnosis of gunshot
injury. Analysis of equivocal fractures by a chemist or ballistic
Funding
analyst trained in elemental analysis can help to differentiate
The author(s) received no financial support for the research,
postmortem taphonomic changes from antemortem lesions.
authorship, and/or publication of this article.
Similarly, the degraded and scavenged bones of animals that
died of pentobarbital toxicosis/euthanasia may still contain
residues of the drug in the skeletal remains. Watterson et al6 References
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2. Haglund WD. Dogs and coyotes: post mortem involvement with human remains.
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still provides a valuable data point in a forensic pathology Human Remains. Boca Raton, FL: CRC Press; 2006:367–381.
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