Pathology in Practice

Laura Polledo AnaPath GmbH, Liestal, Switzerland

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Maria-Gracia De Garnica MicrosVeterinaria, León, Spain

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Roman Schoenauer AnaPath GmbH, Liestal, Switzerland

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Klaus Weber AnaPath GmbH, Oberbuchsiten, Switzerland

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Jacobo Alonso Agrocantabria, Torrelavega, Spain

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Soraya Lolo Veterinary Department, AMSvet Applied Mass Spectrometry Laboratory, Lugo, Spain

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Katie Waine Department of Pathology, School of Veterinary Medicine and Science, University of Nottingham, Leicestershire, UK

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History

A dairy herd of 55 Holstein-Friesian cross cattle from Cantabria, Northern Spain (Atlantic climate) had a sudden onset of severe clinical signs including cardiac arrhythmia. The herd was managed in 2 groups according to the age and stage of production, with 34 adult cows housed and 21 yearlings grazing pasture. Only the housed cows were affected, with 100% morbidity and 7 deaths within 24 hours of the onset of signs in the first cow affected.

Clinical and Gross Findings

On clinical examination, the most striking and consistent finding in all the animals was cardiac arrhythmia, characterized by irregular heart rate, ranging from marked bradycardia (< 30 beats/min) to tachycardia (> 100 beats/min). Dyspnea, dehydration, anorexia, and watery diarrhea were also frequent. All cows showed signs of apathy and had dramatic drops in milk yield, and 12 cows showed neurologic signs, including dullness, reluctance to move, and ataxia. Clinical biochemical analyses that were performed for 3 affected cows showed high concentrations of BUN, serum creatinine, total protein, and albumin and high activities of aspartate aminotransferase (Supplementary Table S1). Finances limited further diagnostic testing in other animals.

Within the first 48 hours, 7 of 34 cows had died or were found moribund and then euthanized. Necropsies from cows 1 and 2 were performed by the attending clinicians at the farm. Both animals showed similar lesions: moderate petechiae and ecchymoses on the serosa of multiple organs in the thoracic and abdominal cavities, including the epicardium (Figure 1) and subendocardium. Severe edema and moderate hemorrhages were detected in the lungs and intestine, and contents throughout the gastrointestinal tracts were very watery and tinged red. Thick, dark green, lanceolate, and leathery leaves (up to approx 25 X 2 cm) were found among partially digested hay in the rumens of both cows. Similar plant debris was also found in the feeders.

Figure 1
Figure 1
Figure 1

Necropsy photographs of the heart with petechiae of the epicardium (A) and of leaves (approx 25 X 2 cm) found in ruminal contents (B) from a 3-year-old Holstein-Friesian cross (cow 1) from a dairy herd that had 100% morbidity and 7 deaths within 24 hours after sudden onset of a range of severe clinical signs, including remarkable and consistent cardiac arrhythmia, among 34 housed adult cows. None of the farm’s 21 yearlings grazing pasture were affected. B inset—An image of fresh leaves from the corresponding plant material.

Citation: Journal of the American Veterinary Medical Association 260, 9; 10.2460/javma.20.06.0319

The leaves were compatible with oleander (Nerium oleander). Withdrawal of the hay lot and treatment of the remaining animals with atropine sulfate (0.06 mg/kg, SC, q 8 h until arrhythmia resolved or for a maximum of 3 days) and supportive IV fluid therapy started promptly. No further animals died, but full recovery of the survivors took 5 to 10 days with a transient loss of body condition (range, approx 10% to 30% decrease) and decreased milk yield (range, 50% to 100% decrease) depending on the individual.

Histopathologic Findings

Samples of the heart, lung, spleen, tracheobronchial lymph nodes, rumen, duodenum, jejunum, ileum, colon, rectum, and kidney from necropsied cows 1 (a 3-year-old cow) and 2 (a 3.5-year-old cow) were routinely processed for histologic examination. Microscopically, lesions were very similar in both animals: hemorrhages and interstitial edema were found in the heart, lungs, and intestinal lamina propria. Within the right and left ventricular myocardium, there were seldom and subtle multifocal clusters of cardiomyocytes that showed hyalinization, irregular shape, and losses of cross striation and nuclei (myocardial degeneration and necrosis) affecting approximately 5% of the myocardium (Figure 2). Cardiac Purkinje fibers multifocally showed karyolysis and karyorrhexis with occasional cytoplasmic fragmentation, associated with edema and hemorrhage in between the Purkinje cells (Figure 3). There was a slight interstitial infiltration of leukocytes in the myocardium.

Figure 2
Figure 2

Photomicrographs of serial sections from a focus of myocardial necrosis in a 3-year-old Holstein-Friesian cross (cow 1) from the herd described in Figure 1. A—Seldom and subtle multifocal clusters of cardiomyocytes show hyalinization, irregular shape, and losses of cross striation and nuclei (myocardial degeneration and necrosis; arrows). There is hypereosinophilia and a slight leukocytic infiltrate. H&E stain; bar = 50 µm. B—Viable cardiomyofibers (asterisk) have red and uniform cytoplasm, whereas damaged cardiomyofibers (arrows) have blue-purple and heterogeneous cytoplasm. Masson trichrome stain; bar= 50 µm. C and D—There is a loss of positive immunolabeling in damaged cardiomyocytes (encircled), in contrast to viable cardiac tissue (asterisks). Anti-muscle actin (C) and anti-desmin (D) immunostainings; bar = 50 µm.

Citation: Journal of the American Veterinary Medical Association 260, 9; 10.2460/javma.20.06.0319

Figure 3
Figure 3

Photomicrographs of the left free ventricular wall from a 3.5-year-old Holstein-Friesian cross (cow 2) from the herd described in Figure 1. A—There is interstitial edema and hemorrhage among Purkinje cell fibers (arrow). H&E stain; bar = 50 µm. B—Purkinje cells have faint nuclei (karyolysis; arrows), cytoplasmic pallor, and the beginnings of cytoplasmic fragmentation. H&E stain; bar = 20 µm.

Citation: Journal of the American Veterinary Medical Association 260, 9; 10.2460/javma.20.06.0319

Masson trichrome stain (MT) and immunohistochemical (IHC) staining against muscle actin (rabbit monoclonal, ab156302, 1:1,000; Abcam Plc) and desmin (rabbit monoclonal, ab32362, 1:1,000; Abcam Plc) were carried out on ventricular myocardial sections (left free ventricular wall, right free ventricular wall, interseptum, papillary muscle) from cows 1 and 2 with the use of adequate validated negative and positive controls. Viable cardiac areas were used as internal staining controls. In damaged areas, the cytoplasm of cardiomyofibers stained blue-purple or heterogeneous mottled colors on MT (Figure 2). Using muscle actin and desmin IHC staining, these necrotic fibers showed loss of immunolabeling and cross-striation pattern, better visualized in longitudinal sectioned fibers. With the use of these techniques, myocardial necrosis was identified in approximately 25% of the cardiomyocytes.

Additional Clinicopathologic Findings

Pooled samples of intestinal content from the jejunum and ileum of cow 1 were submitted for bacterial culture on nonselective and selective media for Clostridium spp and Salmonella spp, in aerobic and anaerobic conditions, to rule out a possible intestinal dysbiosis, and results were unremarkable. Results for detection of clostridial toxins by ELISA on the same intestinal contents from cow 1 were negative for α, β, and epsilon toxins. A pooled sample of ruminal contents and urine from cow 1 was analyzed using high-performance liquid chromatography coupled with tandem mass spectrometry. A highly sensitive and selective multiple reaction monitoring method was set for detection of oleandrin. An enhanced product ion experiment was performed, and the extracted multiple reaction monitoring chromatograms for oleandrin displayed a clear peak at 9.7 minutes (Supplementary Figure S1), which matched for oleandrin, compared against reference spectral library values.

Morphologic Diagnosis and Case Summary

Morphologic diagnosis: multifocal, acute, serosal petechiae and ecchymosis and interstitial edema in the heart, lungs, and intestinal lamina propria. Moderate, multifocal to coalescing, acute, myocardial necrosis with multifocal acute necrosis of cardiac Purkinje cells.

Case summary: oleander intoxication in a dairy herd.

Comments

The acute clinical presentation with 100% morbidity, arrhythmogenic cardiac pulse, and detection of oleander leaves within the rumen and feeders prompted an early withdrawal of the contaminated hay, which is key in the management of this intoxication. Atropine was administered as an antiarrhythmic, with the aim of antagonizing oleandrin vagal activation and increasing heart rate.1 However, atropine or other β-adrenergic agents might increase the risk of tachyarrhythmias, and it has been claimed that they might increase tachyarrhythmic deaths, so the use of this drug is still under debate.2 After the treatment was established, no further animals died, but whether this recovery was due to the atropine itself or to the withdrawal of the oleander-contaminated hay was uncertain. In humans, supportive care and lidocaine, which is the preferred antiarrhythmic, are administered as treatment for oleander poisoning.2 Digoxin-specific antibody fragments against the cardiac glycoside from Digitalis spp have also been proven to be effective for oleander poisoning, as oleandrin has a similar protein structure and the antibodies cross-react.3 However, there is no antidote for cardiac glycosides approved for use in cattle, and digoxin-specific antibody fragments are unaffordable for regular treatments.4 The extralabel drug use of atropine sulfate was performed with owner consent, and the meat and milk withdrawal times were 6 and 28 days, respectively.

Oleander consumption of 50 mg/kg of body weight (between 5 and 10 oleander leaves) may be lethal for an adult cow, with the entire plant being toxic.4 Few cases of oleander toxicosis are reported in cattle, as growing plants are unpalatable, and intoxication is usually associated with accidental ingestion of contaminated hay or silage.5 Within the geographic area where the affected farm was located, oleander was not found growing in accessible fields or the surrounding area. The hay lot supplied to the housed cattle had been first fed the day before clinical signs started. The origin of the hay lot was tracked to a Mediterranean area of Spain (Ebro valley), where this plant is widely distributed.6

Oleandrin causes positive inotropic effects by blocking the heart’s sodium-potassium ATPase with subsequent intracellular retention of Na+ and Ca2+ leading to cardiomyocyte dysfunction.7 At necropsy, the main observations are subendocardial hemorrhages admixed with occasional paler areas in the interventricular myocardium. Pulmonary and intestinal edema and congestion are often observed. Histologically, the myocardial lesions correspond to coagulative necrosis of cardiomyofibers, more prominent in the papillary muscle. If death is acute, these myocardial histologic changes may be very subtle and difficult to detect in H&E-stained tissue sections.8

The remarkable arrhythmia detected in the present report was attributed to the action of oleander cardiac glycosides that cause profound changes in heart contractility. Dyspnea, dehydration, watery diarrhea, anorexia, apathy, and neurologic signs, such as locomotion disturbances and lethargy, have been reported5 for oleander intoxication in cattle. The dyspnea and diarrhea were related to the pulmonary and intestinal edema, respectively, most likely originating from the abnormal cardiac function or direct irritation.9 Neurologic signs were variable and nonspecific, and the origin was not elucidated, as brains were not sampled. Neurologic signs have been suggested to be associated with perivascular and perineuronal edema in oleander intoxication.10

Biochemical values showed a moderate elevation of renal markers. Despite the azotemia detected in our 2 evaluated cases, histologic changes were not evident, most likely due to the acute death with no time for histologic changes to develop.11 The increase in total protein and albumin concentrations was possibly related to dehydration. The high aspartate aminotransferase activity was most likely associated with myofiber damage.12

For the cows of the present report, < 5% of cardiomyofibers had necrotic changes on H&E-stained sections, similar to other reported oleander poisonings.4,8 If death after cardiac insult is rapid, morphological gross and histologic changes may be minimal despite the myocardial dysfunction.4 This fact can make it challenging to diagnose, especially when there is a lack of clinical information. Viable cardiac areas show a uniform red color with MT and a striated pattern when labeled with antibodies against desmin and muscle actin. Necrotic cardiomyofibers turn blueish purple on MT and lose actin and desmin immunolabeling on IHC staining.13 In this case, muscle actin and desmin IHC and MT allowed better detection of damaged fibers, showing that up to 25% of the examined sections of myocardium were affected. These techniques were therefore considered valuable tools for detecting early myocardial ischemia that was not visible with H&E staining, which may be useful to include in veterinary forensic investigations where myocardial necrosis is suspected.

Damage of cardiac Purkinje fibers (conducting fibers) seen in the 2 necropsied cows was a remarkable finding that contributed to the arrhythmia associated with an interruption of impulse propagation.14 Purkinje fibers appear to have a greater sensitivity to digoxin than myocardium (which might be also the case for oleandrin due to their similarities), and cardiac dysfunction might arise first in the cardiac Purkinje fibers by this intoxication.15 This histologic finding has not been previously reported in the literature.

In summary, an acute clinical onset of arrhythmia in multiple animals in a herd should raise the suspicion of cardiac glycoside poisoning regardless of the geographic location of the animals, as the toxin can be ensiled in feed produced in other areas. Clinical detection of marked arrhythmias combined with necropsy examination and careful evaluation of ruminal contents allowed an early diagnosis in this case.

Supplementary Materials

Supplementary materials are posted online at the journal website: avmajournals.avma.org

Acknowledgments

The authors declare that there were no conflicts of interest and that the authors received no financial support.

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