Download Diagnosis of Oleander Poisoning in Livestock

J Vet Diagn Invest 8:3 58-3 64 (1 996)
Diagnosis of oleander poisoning in livestock
Francis D. Galey, Dirk M. Holstege, Konstanze H. Plumlee,
Elizabeth Tor, B ill Johnson, Mark L. Anderson,
Patricia C. B lanchard, Frank B rown
Abstract. S ince mid- 1 989, 3 7 cases of oleander poisoning in livestock have been diagnosed at the California
Veterinary Diagnostic Laboratory S ystem. The most frequent source for oleander exposure was plant clippings.
S udden death was the most common presenting complaint. Other signs reported included diarrhea, pulmonary
edema, tachycardia, cardiac arrhythmias, colic, and lethargy. In the past, a presumptive diagnosis of oleander
poisoning could be based only on matching clinical signs with evidence of consumption of oleander. A new 2
dimensional Thin-layer chromatography analysis of ingesta for oleandrin and an awareness of lesions in heart
muscle have greatly improved the ability to diagnose oleander toxicosis.
Oleander (Nerium oleander) is an ornamental, evergreen shrub. Oleander leaves are leathery and dark
gray-green and have a prominent midrib with secondary veins that are parallel with each other. The plant
is found commonly in the southern United S tates and
most of California. All parts of the plant are toxic.
Ingestion of clippings from oleander is a common cause
of poisoning in animals.
The toxicity of oleander results from several cardiac
glycosides. The most prominent of those glycosides are
oleandrin (oleandrigenin is the aglycone of oleandrin)
and neriine.3 Cardiac glycosides cause poisoning by
inhibiting Na+/K + ATPase. Oleander is an extremely
toxic plant; as little as 0.005% of an animal s body
weight in dry oleander leaves may be lethal (e.g., 1 02 0 leaves for an adult horse).5
Animals exposed to oleander are often found suddenly dead or they present with rapidly developing
nonspecific signs that may resemble colic. When clinical signs are observed, they develop after a delay of
2 -4 hours and may include abdominal pain, weakness,
rumen atony, and excessive salivation.3 ,5,6 Cardiac alterations may include bradycardia or tachycardia, weak
and irregular pulse, heart blocks, and a variety of ventricular arrhythmias. Excitement, intermittent convulsions, dyspnea, and coma may precede death, which
may occur within 2 or as late as 1 2 -3 6 hours after the
onset of signs.
Diagnosis in some lethal cases may be facilitated by
finding leaves in the environment or in the ingesta.
However, leaves may be macerated beyond identification or passed into the posterior gastrointestinal tract.
Thus, definitive diagnosis of oleander poisoning is often difficult. In this report, we describe diagnosis of
oleander toxicosis and several recent cases of oleander
poisoning in California livestock. In addition to correlating signs with exposure to oleander clippings, the
approach to diagnosis described here includes development of a diagnostic test for oleandrin in ingesta and
improved recognition of pathologic lesions in animals
with oleander toxicosis.
Materials and methods
Case histories. S ince mid-1 989, 3 7 separate cases of oleander toxicosis have been confirmed at the California Veterinary Diagnostic Laboratory S ystem. All of the cases originated in the state of California. Cases were selected for this
study based on confirmation of exposure to oleander by identification of the leaves in ingesta or the environment, appropriate signs and lesions, or by chemical identification of
oleandrin. Other possible causes of sudden death, such as
feed additives, nitrate, alkaloids, heavy metals, insecticides,
and appropriate infectious agents, were also investigated. Results from that testing were unremarkable (some findings of
nutritional deficiencies and of unrelated, chronic endemic
disease) and are not reported here.
Pathologic investigations, when performed, followed routine diagnostic laboratory procedures. S amples for histologic
evaluation were fixed in 1 0% neutral buffered formalin and
stained with hematoxylin and eosin (HE) before examination
using a light microscope. Heart sections were obtained from
the papillary muscle region of the left ventricle, right ventricular free wall, interventricular septal region, and, if the
suspicion of cardiac poisoning was high, from atria and auricles.
From the California Veterinary Diagnostic Laboratory S ystem,
University of California, Davis, CA 9561 6 (Galey, Holstege, PlumThin-layer chromatography (TLC) method for oleandrin
lee, Tor, Johnson, Anderson, B rown), and The California Veterinary
Diagnostic Laboratory S ystem, University of California, Tulare, CA assay. S amples (1 0 g) were homogenized with 1 00 ml dichloromethane. Aliquots (2 5 ml) were evaporated to dryness
932 74 (Blanchard).
Received for publication S eptember 5, 1 995.
with a stream of nitrogen at 60 C. The extract was reconsti3 58
Oleander poisoning
Figure 1 . Photomicrographs of left ventricle of the heart from a horse with oleander toxicosis. HE. Top. The subendocardium (S E) is
thickened by edema, hemorrhage, and inflammatory cells. The reaction surrounds Purkinje cells (P) of the bundle of HIS and extends into
the underlying myocardium (M). Bottom. Normal myocytes (M) interrupted by foci of degeneration and necrosis. Note interstitial edema
with fragmentation of myocytes (arrows).
Results
Clinical and pathologic findings. Clinical data are
summarized in Table 1 . The 3 7 confirmed cases of
oleander poisoning resulted in 1 42 deaths out of 2 85
reportedly ill animals. At least 4,504 animals were at
risk (in the same pasture or pen as those affected).
Morbidity ranged from highs of 1 00% to as low as
approximately 1 out of every 1 00 cattle sharing a pasture (exposure of unaffected animals was not assumed,
however). Mortality also varied, although it was as high
as 1 00% of exposed animals in some cases.
Cases occurred throughout the year. Affected animals came from ranches and farms located as far north
as Placer, S onoma, and Y olo counties in Central California, continuing south through S an Diego County.
Cattle were most frequently affected: 2 1 /3 7 reports,
comprising 1 2 2 deaths from 2 62 reported ill, and representing 4,449 of the 4,504 animals at risk. Horses
(8), llamas (6), rheas (1 ), and a big horn sheep (1 ) were
also affected. The sources for the oleander in all of the
cases for which information was available were clippings and/or dry leaves.
For all species, sudden death was the most common
case presentation. Clinical signs, if reported, were nonspecific and included various degrees of colic, diarrhea,
weakness, tachycardia, ataxia, and anorexia. One horse
had ileus, and another had mild colic. Cardiac arrhythmia, which was not specified, was reported for one
llama. Postmortem findings included no lesions for
some peracute cases. The most common lesions observed involved the heart. Gross lesions, when present,
included endocardial hemorrhage associated with increased volumes of pericardial fluid and subepicardial
edema around cardiac vessels. Colon and cecal contents were very fluid in some cases. Edema in the colon
and/or subcutaneous tissues was ocassionaly reported.
Histologically, sites of necrosis included various cardisc regions, although the subendocardium seemed to
be the most common location for lesions (Fig. la).
Typically, the left ventricle was the most severely af-
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Galey et al.
consistent pattern of other spots not found in blanks
or samples spiked with oleandrin alone. Fortification
of diagnostic samples of stomach contents at levels of
5, 0.2 5, 0.1 , and 0.05 ppm all resulted in detection of
oleandrin by TLC. Oleandrin was detected in rumen
contents when spiked at similar levels. Additionally,
oleandrin spikes at 0.1 and 0.02 ppm were identified
in both rumen contents and urine. No spikes at those
levels failed.
Diagnosis in 2 3 of the 3 7 cases benefitted by identification of oleandrin. Chemical analysis of oleandrin
was the only diagnostic evidence for 9 cases. Two additional cases were diagnosed using the chemical test,
with identification of a source of oleander clippings
only after chemical assay suggested the possibility. Rumen and/or stomach contents were the most reliable
diagnostic samples in most cases. However, in 2 instances for horses, cecal or fecal contents had oleandrin, whereas stomach contents had no evidence of the
glycoside. Urine was found to have a trace of oleandrin
in one case, yielding a spot that was similar in intensity
(visual) to a 0.05-ppm spike. Urine had no oleandrin
in 2 other cases for which ingesta was found to have
the toxin.
Discussion
Clinical signs reported for animals with oleander
toxicosis in this study were consistent with those that
Figure 2 . Oleander leaves (2 leaves on the left), which are very
3
toxic, are often mistaken for eucalyptus (gum) tree leaves (2 leaves were previously reported. Those signs, including sudden death, which was most commonly reported, were
on the right). Note distinctive parallel secondary veins in the oleander. B ar = 1 cm.
relatively nonspecific. The evidence of cardiac arrhythmias in live animals also supports toxicosis due to a
cardiac glycoside such as oleandrin. Many animals had
fected. Lesions also were present in the walls of other no lesions or were too severely autolyzed to be assessed
portions of the heart, including the auricles. Those pathologically. When lesions were found, they were
lesions in the heart included subendocardial hemor- consistent with gastroenteric and myocardial damage.
rhages and multifocal myocardial edema, degenera- The pulmonary edema and liver damage were most
tion, and necrosis (Fig. lb). Pulmonary edema, mild likely secondary to the cardiac alterations, leading to
hepatic congestion, and lympholysis were also report- congestion in those organs.
ed. Although diarrhea and gastrointestinal upset were
All of the cases for which we obtained an adequate
reported clinically, lesions in the gut were minimal.
history involved exposure to clippings. This finding is
Diagnosis. Exposure to oleander in many of the consistent with reports that oleander is apparently not
cases was indicated by identification of the plant leaves palatable to animals in the form of fresh leaves.3 The
in ingesta and/or the environment. The leaves must continued high toxicity of dried/wilted material is also
be distinguished from those of other forbs, most com- supported. These findings suggest that oleander toximonly those of Eucalyptus (gum) trees (Fig. 2 ). Ole- cosis in livestock is more prevalent than originally
ander is characterized by an oblong leaf shape, typical thought or perhaps is increasing in incidence. Although
parallel secondary veins, and characteristic stomata reports of oleander toxicosis in livestock increased
(visible under a dissecting microscope).
slightly from 1 989 to 1 994, a large increase in diagDiagnosis in the most recent cases was supported by noses occurred in 1 994 and 1 995. The increase most
identifying oleandrin using TLC (Fig. 3 ). Two-dimen- likely resulted from increased awareness of the disease,
sional TLC was the best method of isolating the olean- alertness to possible lesions, and the availability of a
drin. In addition to the oleandrin, chromatography of chemical test for oleandrin.
Radioimmunoassay for digoxin, which may crosssamples of leaves and fortified ingesta also revealed a
Oleander poisoning
3 63
Figure 3. Two-dimensional thin-layer chromatograms used for diagnosis of oleander toxicosis. S pots on bottom and top right edge are
oleandrin standards. a. B lank chromatogram. b. Oleander plant material. c. Rumen content from healthy animal spiked with oleandrin
(0.05 ppm). d. Colon content from animal with oleander poisoning. Note pattern of spots below the oleandrin spot for the plant and
naturally exposed animal.
react with oleander, has been suggested as a diagnostic
test.1 ,7 However, samples of animal urine that were
spiked with oleander glycosides at 0.01 ppm did not
yield a positive result in at least 1 assay. Therefore,
development of a different approach to chemical identification of oleander toxicosis was desired. The method developed in our laboratory has been reliable and
has allowed identification of cases in which history
subsequently revealed exposure to oleander (e.g., the
miniature horses in 1 994).
The metho d itself is repeatable and relatively
straightforward to conduct. Lipids present in the extract do not dissolve in methanol but do dissolve in
less polar solvents such as dichloromethane. The pu-
rified extract should be reconstituted to volume in
methanol to reduce matrix interferences on the TLC
plate. The first development in dichloromethane helps
resolve the oleandrin from the residual lipids, yet
oleandrin does not migrate during this step. The sensitivity of the method is high; it can detect as little as
0.02 ppm of oleandrin in ingesta and urine. The method detection limits that we use clinically are 0.05 ppm
for ingesta and 0.02 ppm for urine. In addition to the
ability of the method to identify the oleandrin as a
marker toxin, natural exposures are also supported by
evidence of other constituents of oleander in diagnostic
samples (Fig. 3 ). Those spots (below the oleandrin spot)
are likely to be some of the other toxic cardiac gly-
3 64
Galey et al.
cosides in the plant, one of which is probably oleandrigenin, the aglycone of oleandrin.5
B ased on sudden death, often with no (or very nonspecific) signs, oleander must be differentiated from a
variety of poisons such as other toxic plants,2 industrial
poisons, and some feed additives. The diagnosis is
often aided by identifying the plant in the environment
or ingesta, but if the animal has survived for any length
of time, the plant may no longer be evident. Identifiable leaves were more likely to have been found in
cattle rumens, whereas intact leaves was less commonly found in for horses and llamas. Additionally,
oleandrin was found in cecal or fecal material from
some horses when it was absent in the stomach contents. Thus, this work suggests that diagnosis of oleander toxicosis for animals, especially horses, that survive several hours or longer is very much helped by
testing of cecal or fecal material for oleandrin.
Incidences of oleander poisoning diagnosed by this
laboratory has risen. A history of exposure to leaves
in an animal that is depressed, has colic, or has suddenly died is suggestive of oleander poisoning. Large
amounts of fluid in the pericardial cavity, cardiac hemorrhage, pulmonary edema, and gastroenteritis are also
consistent with oleander toxicosis. Other plant toxicants such as alkaloids must also be ruled out.4 Identification of oleandrin in the ingesta, especially from
the posterior gut (cecal content and feces), provides the
evidence of exposure needed for more definitive diagnosis. This chemical test for oleander has much improved our ability to diagnose oleander poisoning in
livestock.
Acknowledgements
We thank Terry Wildman and B ob Nordhausen for photographic assistance. Analytical method development was
supported in part by the California Center for Equine Health
and .Performance and the Livestock Disease Research Laboratory.
Sources and manufacturers
a. Mycochar fi , Romer Laboratories, Columbia, MO.
b. Uniplatefi silica gel HL, 2 50 m, Analtech, Newark, DE.
c. S igma Chemical Co., S t. Louis, MO.
d. Toxi-lab heated warmer, Toxi-Lab, Irvine, CA.
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