CASE I: N-697-15 (JPC 4085531)
Signalment:
10-year-old, female, Crioula, horse (Equus caballus)
History:
This is one horse out of a group of 15 horses of the same breed held together exclusively on a native pasture without feed supplementation. Five adult horses got sick with a common history of neurological signs and progressive loss of weight ? three adult horses and one colt died after a clinical course of 90 days and the surviving horse was euthanized for humanitarian reasons. One of the horses was necropsied. Clinical signs were characterized by progressive weight loss, incoordination, stiff gait, aimless walking, abnormal behavior, and hyperesthesia with locomotion difficultly when provoked to move. According to the owner, affected horses were found trapped in fences or in dense bushy areas. A heavy infestation of broomweed (Sida carpinifolia) was observed in the pasture where the 15 horses were held, mainly in shady places close to fences and riversides. All horses were vaccinated for rabies.
Gross Pathology:
Necropsy findings in the horse of this report included bilateral extensive lacerations of the skin of distal portion of hind limbs and poor body condition. The large colon was moderately distended by fecal content and there was moderate urinary bladder repletion.
Laboratory Results:
Fresh samples of brain and spinal cord were submitted to direct FAT for rabies with negative results.
Microscopic Description:
Histologically, neurons and astrocytes from telencephalic cortex, diencephalon, striate body, rhombencephalon, cerebellum and spinal cord (cervical and lumbar intumescences, and thoracic and sacral segments) contain varying degrees of swollen and fine vacuolated cytoplasm giving the cells a foamy appearance. The lesions were specially marked in thalamus, hippocampus, cerebellum and in all examined levels of the spinal cord. Occasionally, there were neurons with shrinking and hypereosinophilic cytoplasm (neuronal necrosis), and multifocal axonal spheroids. There was also moderate astrogliosis of the Bergmann glia which was highlighted by the GFAP IHC. In several ganglia (mesentery celiac, trigeminal and paraspinal) there was mild neuronal cytoplasmic vacuolization.
Sections of cerebellum, pons, thalamus, and hippocampus were submitted for lectin histochemistry technique with commercial lectins (Vector Laboratories, Burlingame, CA). Immunohistochemistry (IHC) to detect anti-glial fibrillary acidic protein (GFAP) was performed in adjacent sections using streptavidin-biotin-peroxidase complex, with anti-glial fibrillary acidic protein (GFAP) at 1:500 and anti-S100 at 1:200 as primary antibodies. Antigens were retrieved through heat and reaction was revealed with 3?3-diaminobenzindine (DAB) chromogen (DAKO). As negative control, a brain section from a normal age-matched horse was used.
Contributor?s Morphologic Diagnoses:
Neuronal vacuolar degeneration, moderate to severe, diffuse, chronic.
Contributor?s Comment:
The diagnosis of Sida carpinifolia poisoning in these cases was based on clinical, epidemiological, histopathological, and lectin histochemistry findings.
Normal cellular catabolism directs a steady stream of endogenous complex macromolecules into vesicular compartments (lysosomes) for degradation to simple molecules that may be reused or excreted. These essentially autophagic pathways are the means through which a cell recycles its own constituents. Storage diseases occur when there is accumulation of macromolecules in a cell which has compromised intracellular mechanisms for digestion, disposal, or transport. Most storage diseases involve neurons and translate as neurologic impairment5 although other populations of stable or permanent cells may be affected. Lysosomes are the kingpins of the degradation mechanism for intracellular digestion of macromolecules. Lysosomal
digestion can be impaired in several ways. The most relevant is the deficient activity of a specific lysosomal hydrolase due to a genetic defect or to an acquired condition which compromises one or more of the lysosomal enzymes.5
Acquired lysosomal diseases in domestic animals are usually, but not always, induced by the ingestion of plants containing swainsonine, including Swainsona spp. in Australia, Astragalus spp. and Oxytropis spp. in South and North America, China, and Africa,9,14,17 Ipomoea carnea subsp. fistulosa,2 Ipomoea sericophylla and I. riedelii,3 Ipomoea verbascoidea,16 Turbina cordata,8 and Sida carpinifolia10,11,15 in Brazil.
S. carpinifolia (Malvaceae) is a perennial shrub, frequently found in humid and shady areas of Brazil in the southern, southeast and midwestern regions. It is also known by the synonyms S. acuta Burm., S. ulmifolia Mill., S. acuta var.carpinifolia (L.f.) K. Schum, and S. frutescens Cav. The plant is an erected perennial shrub 30-70 cm high. The leaves are alternate and short-peciolate, with an upper glabrous surface and a lower surface with sparse hair over the veins. The flowers are distributed singly or in small clusters and are yellow with seven or eight carpels. The ingestion of S. carpinifolia induces an intracellular storage of oligosaccharides caused by indolizidine alkaloid (swainsonine),10 which causes inhibition of lysosomal α-mannosidase and α-mannosidase II from Golgi complex,18 affecting typically neurons.17 It is a chronic disease with clinical signs characterized by neurological disturbances. The toxicosis is also associated with abortions and stillbirths.10
Several species of grazing livestock and wildlife such as goats,10 sheep, 22 cattle,11 horses15 and fallow deer19 have been described to be affected by S. carpinifolia poisoning.
Lysosomal storage diseases, both genetic and acquired, are uncommon in horses, but there are reports of inherited neuronal ceroid lipofuscinosis23 and acquired cases of poisoning by Astralagus spp. and Oxytropis spp. in North America and Swainsona canascens var horniana in Australia.14
The clinical findings in ponies naturally poisoned by S. carpinifolia were described15 and include stiff gait, general muscular tremors, colic, rolling, moans, recumbence and death 15-20 days after introduction into an area with large amounts of S. carpinifolia. The affected horses of this report had abnormal behavior with exacerbated reactions and difficult locomotion after induced movement, incoordination, stiff gait and aimless walking. Affected horses were frequently found dead trapped in fences or in dense bush areas.
It is possible that signs of colic in these cases result from neurogenic motility dysfunction of the large intestine due to lesion in the celiac ganglia and submucosal and myenteric plexuses.
Abnormalities in coordination and behavior are caused, mainly, by chronic neurodegenerative lesions triggered by the toxic active principle of the plant, which leads to oligosaccharides storage in the cytoplasm of cells from multiple tissues.18
Pathogenesis involves a deficiency of the lysosomal enzyme α-mannosidase, which is responsible for the catabolism of multiple glycoprotein portions; thus, it inducing the lysosomal storage of a wide range of oligosaccharides.13 The disease induced by the plant toxic principle differs from the inherited condition, since the former causes also the inhibition of mannosidase II produced in Golgi complex in addition to inhibition of α-mannosidase.5 Experimentally it has been shown that the clinical evolution and the clinical signs are related with the duration of ingestion and with the amount of the plant that was consumed.10,22 The clinical course of the horses of this report was 90 days, different from what was described in ponies, where the toxicosis showed a peracute to acute course.
The necropsy findings in cases of S. carpinifolia poisoning are not significant.10,11,22 In the current case, there was only moderate distension of large colon by fecal content, which was also described in ponies poisoned by S. carpinifolia.15 Microscopic lesions of brain and spinal cord were mainly characterized by marked cytoplasmic vacuolization of neurons and astrocytes, with few necrotic neurons and axonal spheroids; moderate proliferation of Bergmann astrocytes in cerebellum, and moderate vacuolization of thyroid follicular epithelial cells and proximal convoluted tubular cells, and mild vacuolization of hepatocytes. These were similar to what has already been described in affected cattle,11 sheep,22 goats,10 and ponies.15 Others have also described cytoplasmic vacuolization of acinar pancreatic epithelial cells.10,11,19,22
Lectins are proteins or glycoproteins of non-immune origin that bind reversibly to specific residues of carbohydrates (glycoproteins, glycolipids and glycosaminoglycans).10 The lectin histochemistry analysis may detect certain substances in a tissue where a lesion has occurred and helps to establish a correlation between its presence, quantity, severity and extension of the process. The efficiency of this analysis has been already demonstrated several studies through the detection of complex substances containing sugars, related or not with storage diseases, such as glycoproteinosis10 and glicolipidoses.1 The brain sections of the horse of this report submitted to this type of analyses revealed moderate to marked cytoplasmic stain in neurons when Triticum vulgaris (WGA) and Succinyl Triticum vulgaris (sWGA) were employed which indicates the expression of ß-D-N-acetylglucosamine and acetylneuraminic acid. Similar observations were made when Concanavalia ensiformis (Con-A) was employed, which is a specific lectin for α-D-mannose α-D-glucose. The mild to moderate stain for UEA I, PNA and SJA lectins observed in the horse of this report has been described in goats poisoned by the same S. carpinifolia, as well as in tissues without lesions.10 IHC anti-S100 has highlighted the
increasing number of Bergmann astrocytes in cerebellum, in addition to a higher expression of GFAP. The Bergmann?s glia is a specialized population of astrocytes that occurs in the Purkinje cell layer of cerebellum.4 The increased numbers of Bergmann astrocytes has been described in cases of primary loss of Purkinje cells with secondary depletion of the molecular and granular layers and in poisonings by plants containing swainsonine.
Contributing Institution:
Setor de Patologia Veterinária, UFRGS, http://www.ufrgs.br/patologia/
JPC Diagnosis:
Diencephalon with hippocampus and thalamus, neurons and glia: Cytoplasmic vacuolation, diffuse, marked with rare neuronal necrosis, satellitosis, and spheroids.
JPC Comment:
The contributor provides a thorough review of the pathogenesis of chronic swainsonine ingestion, as seen in this case associated with broomweed (Sida carpinifolia) ingestion. Molecularly similar to the simple sugar mannose, swainsonine inhibits both lysosomal enzyme α-mannosidase and golgi mannosidase II, resulting in an inability affected cells to to process mannose rich oligosaccharides, which subsequently accumulate in lysosomes and cause cellular dysfunction.6,20
As noted by the contributor, swainsonine containing plants naturally grow in several continents and poison significant numbers of livestock. In the United States, plant species in genera Astralagus and Oxytropis are colloquially known as locoweeds, with swainsonine commonly referred to as "loco-toxin" and its associated neurologic clinical condition is known as "locoism". Astralagus and Oxytropis are both extensive genera, with 354 species and 198 varieties of Astralagus reported in the United States and Canada while there are 22 species and 35 varieties of Oxytropis.20
Identified as an issue by stockmen in the United States as early as 1873, locoism posed such a significant problem that a field station was established and dedicated to its study in Hugo, Colorado in 1905. Subsequent research lead to the publishing of a USDA bulletin titled "The locoweed disease of the plains" by C.D. Marsh in 1909. Interestingly, Marsh connected locoism with a similar condition in Australia known as "peastruck", which was associated with consumption of the Darling Pea (Swainsonia spp.). Seventy years later, Colgate et. al. isolated and described the toxin indolizidine alkaloid swainsonine from Swainsonia canescens in Australia. The toxin was subsequently isolated from Astralagus and Oxytropis in 1982.20
In Brazil, two recent studies have described antemortem techniques in regard to the presumptive diagnosis of swainsonine toxicity.6,21 The first evaluated blood smears from guinea pigs fed 50 grams of I. marcellia per day. A repeatable finding after only five days of ingestion was the cytoplasmic vacuolation of lymphocytes in blood smears, predominantly at the periphery of the cell.12 Another study evaluated the usefulness of percutaneous hepatic biopsies using Menghini needles in goats fed 4g/kg/day of dried I. marcellia (0.8mg/kg/day swainsonine). Hepatocellular vacuolization consistent with lysosomal storage disease was identified in all affected goats starting at day seven. Sections were subsequently labelled using lectin histochemistry staining with Concanavalia ensiformis (CON-A) and Triticum vulgaris (WGA).21
Swainsonine producing fungal symbionts, such as Undifilum spp., have been found to be associated with multiple plant species associated with swainsonidosis, including Astragalus mollissimus, Oxytropis lambertii, and O. sericea in North America4, Astragalus and Oxytropis in China, and S. canescens in Australia.5 Furthermore, swainsonine content has been found to correlate with the degree of endophyte infection, suggesting endophytes at least partially influence the degree of toxicity.4
References:
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