Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
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- 3 62 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. References 1 . Ansford A, Morris H: 1 983, Oleander poisoning. Toxicon Suppl. 3 :1 5-1 6. 2 . Cheeke PR, S hull LR: 1 985, Natural toxicants in feeds and poisonous plants. AVI, Westport, CT. 3 . Everist S L: 1 981 , Apocyanaceae. In: Poisonous plants of Australia, 2 nd ed., pp. 77-89. Angus and Robertson, London, England. 4. Holstege DM, S eiber JN, Galey FD: 1 995, Rapid multiresidue screen for alkaloids in-plant material and biological samples. J Agric Food Chem 43 :691 -699. 5. Kingsbury JM: 1 964, Poisonous plants of the United S tates and Canada, pp. 2 64-2 67. Prentice-Hall, Englewood Cliffs, NJ. 6. Mahin L, Marzou A, Huart A: 1 984, A case report of Nerium oleander poisoning in cattle. Vet Hum Toxicol 2 6:3 03 -3 04. 7. Osterloh J, Herold S , Pond S : 1 982 , Oleander interference in the digoxin radioimmunoassay in a fatal ingestion. J Am Vet Med Assoc 2 47:1 596-1 597.