Signalment:  

Adult merino sheep of unidentified gender, (Ovis orientalis aries) ovineThis animal was one of multiple affected animals from a flock in Narrogin, Western Australia, submitted in March 2009. The animals had been turned out onto a lupin and wheat paddock (presumptive stubble) approximately six weeks previously (January 2009). Multiple animals were displaying signs of marked lethargy and weakness. Ten sheep were recumbent and unable to rise and were subsequently euthanized.

Gross Description:  

A post mortem examination was conducted on one of the affected animals in the field by the local veterinarian, who subsequently submitted both fresh liver and formalin fixed tissues to the Western Australian Department of Agriculture and Food for examination. The veterinarian reported extremely dry ruminal contents (suggesting dehydration) and the presence of peritoneal and pleural proteinaceous effusions. The liver was reportedly small with a yellow tinge and the kidneys appeared very dark.

Histopathologic Description:

The hepatic architecture is diffusely disrupted by increased fibrous tissue surrounding portal triads, with fine septa ramifying outwards, distorting the lobular architecture and dissecting the hepatic plates resulting in isolation of small clusters of hepatocytes with occasional individualisation. Numerous hepatocytes appear swollen with moderate to marked anisocytosis and there are numerous mitotic figures, many of which appear abnormal, often arrested in metaphase. There are occasional scattered shrunken hypereosinophilic hepatocytes with condensed nuclei, consistent with apoptosis. Mild, diffuse bile duct proliferation is also apparent. Low numbers of lymphocytes, macrophages and neutrophils are present, particularly periportally. Modest numbers of canaliculi contain bile plugs. Also scattered throughout the hepatic parenchyma are occasional cells (hepatocytes and Kupffer cells +/- macrophages) with faint intracytoplasmic yellow-golden brown granular pigment. Shikatas Orcein and Schmorls stains demonstrated this pigment to be a mix of copper and lipofuscin (+/- ceroid), respectively. A Perls Prussian blue also demonstrated the presence of a minimal amount of iron.

Morphologic Diagnosis:  

Liver: Moderate, diffuse, chronic hepatocellular dysplasia and dissecting portal fibrosis, with mild cholestasis, biliary hyperplasia and intracytoplasmic pigment accumulation

Lab Results:  

Aerobic and anaerobic culture performed on the samples of fresh liver yielded no growth.

Condition:  

Phomopsin toxicosis (lupinosis)

Contributor Comment:  

The history, clinical signs, reported necropsy findings and histopathological changes observed in the liver of this sheep are consistent with the syndrome of chronic phomopsin poisoning, which is also widely referred to as lupinosis. Lupinosis is a mycotoxicosis seen in animals ingesting lupin stubble or seed (Lupinus spp.) infected with the fungus Diaporthe toxica (anamorph Phomopsis sp.). Diaporthe woodii (anamorph Phomopsis leptostromiformis) was originally (and incorrectly) thought to be the source of the causative toxins.(6, 11) Diaporthe toxic is parasitic on green lupins, and grows saprophytically on the dead host,(2, 9) and produces a number of different toxins, the best known of which are the phomopsins A, B & C.(8) These compounds are hexapeptides, and appear to act predominately on the hepatic parenchyma, where they bind to tubulin and inhibit microtubule polymerization leading to progressive loss of microtubules. This in turn disrupts mitosis, resulting in mitotic arrest in metaphase and commonly fatty infiltration.(1) It is important to note that this disease is a distinct entity from the disease known as lupine poisoning, which causes acute neurological signs and results from the ingestion of lupines containing quinolizidine alkaloids.(7, 9) Lupinosis most commonly occurs in sheep, but also occurs in cattle, and, rarely, in horses and pigs.(4, 7) Outbreaks of the disease are most common in countries where lupins are eaten as a dead standing crop or grazed as stubble, a situation most often seen in Australia. Lupinosis occurs occasionally, but not commonly, in South Africa and New Zealand. (4)

Clinically, a variety of clinical syndromes are observed, dependent on the species involved, the dose of toxin and the duration of exposure.(2, 6, 8) In sheep, acute lupinosis occurs when animals graze highly toxic stubbles over a short period. Clinical signs include anorexia, lethargy, weakness and jaundice. Rarely, hepatic encephalopathy or photosensitization is observed. Clinical signs are typically seen within two days of introduction to stubble. Most animals within a flock are affected and deaths occur within three or four days.(7) Chronic lupinosis occurs following the ingestion of low doses of toxins over a prolonged period. Variable numbers of sheep within a flock are affected and animals are typically weak and in poor body condition. Jaundice may or may not be apparent, and those sheep that are jaundiced are commonly anemic. Mortality rates are low. The disease course may also be subacute and intermediate in severity, and it is this form of the disease that is most commonly observed in Australia.(8) A toxinrelated nutritional myopathy has also been reported in association with lupinosis, which may or may not respond to supplementation with vitamin E or selenium.(8) Experimental phomopsin toxicity has been shown to reduce reproductive performance also.(7)

On gross examination, livers from acutely affected sheep are enlarged and frequently discolored yellow or tan, with histological examination revealing variable degrees of hydropic change and an increased rate of hepatocyte loss (from widespread apoptosis). The degree of fatty change is variable and depends largely on the nutritional status of the animal. As the disease course progresses, increased, but ineffective mitotic activity becomes evident with the presence of numerous arrested mitoses. Progressive hepatic fibrosis occurs and there is accumulation of complex granular pigment within macrophages in the affected tissue, and may include any combination of copper, ferric iron, ceroid or lipofuscin. Variable degrees of portal hyperplasia may also be present.(6, 9) Some of these changes, including hepatic fibrosis and biliary hyperplasia, can also be observed in other hepatotoxicities including pyrrolizidine alkaloid and aflatoxicosis. As in phomopsin toxicity, these toxins inhibit normal mitosis, and DNA replication frequently continues resulting in the production of greatly enlarged hepatocytes (megalocytosis). In Australia, sheep grazing lupin stubble may also be concurrently exposed to plants containing pyrrolizidine alkaloids such as heliotrope (Heliotropium europium), which may result in additive or synergistic hepatotoxity.(2)

Lupinosis has been largely eliminated in Australia by the breeding of Phomopsis-resistant lupins, and is seen now in years with late finishes and/or increased summer rainfall.

JPC Diagnosis:  

Liver: Hepatocellular degeneration and necrosis, diffuse, moderate, with fibrosis, intracanalicular cholestasis, Ito cell hyperplasia, and prominent hepatocellular mitotic activity.

Conference Comment:  

A few other noteworthy histologic features in this case include a decrease in the lobule size and a decrease in the distance separating central and portal areas, reflecting hepatocyte necrosis and atrophy. Other potential toxic causes for this lesion would include aflatoxin, which may present with more hepatic regeneration, bridging portal fibrosis, biliary hyperplasia and hemorrhagic necrosis, with maintenance of hepatic trabeculae; sporidesmin, a mycotoxin which targets biliary epithelium and causes necrosis; microcystin, a toxin of blue-green algae, which causes disassociation of centrilobular hepatocytes and submassive hepatic necrosis and hemorrhage; and pyrrolizidine alkaloids, which also cause bridging fibrosis, biliary hyperplasia, and megalocytosis(9). Sheep appear to have increased resistance to pyrrolizidine alkaloid intoxication compared with other species.

The contributor mentioned the possibility of photosensitization in acute phomopsin toxicosis. Photosensitization appears as erythema, dermal edema, vesicle or bullae formation, exudation and extensive epidermal necrosis. There are three types of photosensitization. Type I, or primary photosensitization, occurs from ingestion of a plant or drug containing photoreactive substances, which are deposited in the skin. Type II photosensitization occurs in animals with a genetic inability to metabolize heme pigments, which results in a buildup of the photoreactive hematoporphyrin pigments, such as uroporphyrin I, coproporphyrin I, and protoporphyrin III. Type III, or hepatogenous photosensitization, occurs with an abnormal buildup of phylloerythrin, which is a degradation product of chlorophyll, because of a damaged or immature liver. The photosensitization in this case is type III and is secondary to cholestasis(3). In cattle, phomopsin toxicity causes anorexia and ketosis in pregnant or lactating cows, and chronic exposure leads to fibrotic hepatitis with nodular regeneration (cirrhosis)9.

The toxic principles of true lupinosis (not to be confused with phomopsin toxicosis) are quinolizidine alkaloids, which causes nicotinic effects such as salivation, ataxia, seizures, and dyspnea, and the alkaloid anagyrine, which causes arthrogryposis in calves and lambs following in utero exposure(5, 10).

References:

1. Allen JG: Recent advances with cultivated lupins with emphasis on toxicological aspects. In: Poisonous Plants: Proceedings of the Third International Symposium, ed. James LF, Keeler RF, Bailey EM, Cheeke PR, Hegarty MP, pp. 229 233. Iowa State University Press, Ames, IA, 1992
2. Cheeke PR: Toxins in protein supplements and grain legumes. In: Natural Toxicants in Feeds, Forages, and Poisonous Plants, 2nd ed, pp. 163 193, 1998
3. Ginn PE, Mansell JEKL, Rakich PM. Skin and appendages. In: Maxie MG, ed. Jubb, Kennedy and Palmers Pathology of Domestic Animals. vol. 1, 5th ed. Philadelphia, PA: Elsevier; 2007:623-6.
4. Kellerman TS, Coetzer JAW, Naud+�-� TW: Plant poisonings and Mycotoxicoses of Livestock in Southern Africa, pp. 27-29. Oxford University Press, Cape Town, 1988.
5. Maxie MG, Youssef S. Nervous system. In: Maxie MG, ed. Jubb, Kennedy and Palmers Pathology of Domestic Animals. vol. 1, 5th ed. Philadelphia, PA: Elsevier; 2007:247.
6. Peterson JE, Jago MV, Payne AL, Stewart PL: The toxicity of phomopsin for sheep. Aust Vet J 64: 293 298, 1987
7. Radostits OM, Gay CC, Done SH, Blood DC, Hinchcliff KW, Constable PD: Diseases associated with toxins in plants, fungi, cyanobacteria, plant-associated bacteria, and venoms in ticks and vertebrate animals, 10th ed., pp.1906 1908. Saunders, Philadelphia, PA, 2007.
8. Seawright AA: Mycotoxins and Mycotoxicoses. In: Animal Health in Australia: Chemical and Plant Poisons, vol. 2, 2nd ed., pp. 173 177. Australian Government Publishing Service, Canberra, 1989
9. Stalker MJ, Hayes MA: Liver and Biliary System. In: Jubb, Kennedy and Palmers Pathology of Domestic Animals, vol. 2 ed. Maxie, MG, 5th ed., pp. 370-6. Elsevier, Philadelphia, PA, 2007
10. Thompson K. Bones and joints. In: Maxie MG, ed. Jubb, Kennedy and Palmers Pathology of Domestic Animals. vol. 1, 5th ed. Philadelphia, PA: Elsevier; 2007:61.
11. Williamson PM, Highet AS, Gams W, Sivasithamparam K, Cowling WA: Diaporthe toxica sp. nov., the cause of lupinosis in sheep. Mycol Res 98: 1364 1368, 1994

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3-1. Liver

3-2. Liver

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