Signalment:  

10-12-month-old lambs (breed unknown), sex unknown, Ovis ariesAbattoir liver specimens derived from 260 lambs from New South Wales. Fifty percent of the livers were condemned at the abattoir. Lambs had no previous history of illness.

Gross Description:  

Liver had retained their shape but were small, yellow and the capsular surface was markedly granular in appearance. On cut section, nodular regeneration was apparent throughout.

Histopathologic Description:

The capsular surface of the liver was undulated. The normal acinar architecture of the liver was replaced by extensive nodular regeneration and segmental collapse and condensation. The portal triads showed increased mature biliary ductular profiles and there was a moderate mononuclear, primarily lymphocytic infiltration, which extended into the surrounding sinusoids. A moderate degree of fibrosis was present, radiating from the portal triads into the periphery of the regenerative nodules. 

Periportal hepatocytes appeared large, and many appeared to be entrapped by collapsed stroma. The nuclei varied in sizes and shape, but were predominantly large and vesicular with dispersed chromatin. Other nuclear profiles included dark nuclei with smudged nuclear chromatin, fibrillary nuclear chromatin, and irregularly shaped nuclei.

There was a minimal degree of fatty change affecting hepatocytes within regenerative nodules. (Note: The degree of fatty change present may vary between submitted slides). The majority of nuclei within these nodules were unremarkable. Occasional apoptotic hepatocytes were scattered throughout the parenchyma.

Morphologic Diagnosis:  

Chronic active hepatopathy with marked nodular regeneration, hepatic magalocytosis and karyomegaly, liver sheep.

Condition:  

Pyrollizziddine alkaloid toxicosis

Contributor Comment:  

The histological changes present are indicative of a two-phase process. Previously there has been extensive loss of hepatocytes, resulting in condensation of parenchyma and liver shrinkage accompanied by nodular regeneration. Currently, residual periportal hepatocytes are undergoing degenerative changes with megalocytosis, karyomegaly and apoptosis.

Anecdotal history suggested that these lambs had been supplemented prior to slaughter with lupin grain. In addition, it is likely that these animals had been recently grazing lupin stubble.

Lupinosis is a sporadic disease reported primarily in Australia, New Zealand, South Africa and Europe.⁴ It is a mycotoxic liver disease caused by infection of Lupinus spp with the fungus Diaporthe toxica (formerly Phomopsis leptostromiformis). In southern regions of Australia, Lupinus spp (primarily L. cosentini) are commonly used as fodder, either as stubble or as grain.⁴ The fungus produces the toxic agents phomopsin A and B with A being two to three times more toxic than B.³ These toxins bind to tubulin and interfere with the ability of hepatocytes to form microtubules and therefore undergo mitosis. The result is hepatic atrophy and fibrosis. Hepatocytes typically swell and have large vesicular nuclei.

Lupinosis is typically a sub-acute to chronic disease, and can affect other species including cattle, donkeys, goats, horses and pigs.¹ Clinically sheep show non-specific neurological signs and frequently die from misadventure or from copper poisoning. Other organ systems can exhibit cytotoxic effects, including adrenal glands, pancreas, kidneys, rumen and skeletal and cardiac musculature.¹

Lupinus spp themselves also contain quinolizidine alkaloids that can cause teratogenic abnormalities such as crooked calf disease (due to angyrine) and neurotoxic signs.⁵

Although the history and pathological changes present in this case are suggestive of lupinosis, other causes of toxic hepatophy including pyrrolizidine alkaloids cannot be excluded.

JPC Diagnosis:  

1. Liver: Nodular regeneration, diffuse, with megalocytosis, biliary reduplication, and moderate portal bridging fibrosis, breed unspecified (Ovis aries), ovine.
2. Liver: Hepatitis, lymphocytic, subacute, multifocal, mild.

Conference Comment:  

Conference participants suggested a differential diagnosis of lupin toxicosis, pyrrolizzidine alkaloid toxicosis and aflatoxicosis as potential causes of the changes noted in the distributed slides.

Phomopsis leptostromiformis, a fungus that grows on lupine (Lupinus sp.) plants, produces a toxic metabolite, phomopsin. Affected livers exhibit multifocal necrosis and remaining hepatocytes undergoing mitotic arrest in metaphase, resulting in a marked increase in mitotic figures. ² Chronic affected livers are smaller than normal as a result of necrosis, inability to regenerate due to mitotic inhibition, and progressive fibrosis.⁵ Nodular regeneration may occur with sporadic ingestion of the toxin.⁵

Following ingestion, pyrrolizidine alkaloids are converted to pyrrole esters by hepatic cytochrome p450 enzymes, which react with cytosolic and nuclear proteins and nucleic acids to inhibit DNA synthesis and mitosis in hepatocytes.⁵ Megalocytosis, a characteristic finding in pyrollizidine alkaloid toxicosis, occurs when some hepatocytes are able to replicate their DNA yet are unable to divide.⁵

Aflatoxins are also metabolized by the hepatic mixedfunction oxidase system to toxic and non-toxic metabolites. ⁵ The most potent of these is the 8,9-epoxide metabolite of aflatoxin B1, which binds to adenine in nucleic acids, resulting in very similar microscopic findings to animals metabolizing pyrollizzidine alkaloids.⁵

Chronic inconsistent ingestion of any of these toxic principles can result in end-stage liver disease over time. The characteristic micro- and macronodular regeneration seen in end-stage livers can have numerous causes other than toxicity: ²

1. Chronic toxicity (therapeutic agents or naturally occurring toxins)
2. Chronic cholangitis and/or obstruction
3. Chronic congestion (right side heart failure)
4. Inherited disorders of metal metabolism (copper or iron)
5. Chronic hepatitis
6. Idiopathic

References:

1. Allen JG: Proceedings No. 103 Veterinary Clinical Toxicology, pp. 113-131. The Post-Graduate Committee in Veterinary Science, The University of Sydney, Sydney, Australia, 1987
2. Cullen JM: Liver, biliary system, and exocrine pancreas. In: Pathologic Basis of Veterinary Disease, eds. McGavin MD, Zachary JF, 4th ed., pp. 413, 444-446. Elsevier, St. Louis, MO, 2007
3. Culvenor, CCJ, Beck AB, Clarke M, Cockrum PA, Edgar JA, Frahn JL, Jago MV, Lanigan GW, Payne AL, Peterson JE, Petterson DS, Smith LW, White RR: Isolation of toxic metabolites of Phomopsis leptostromiformis responsible for lupinosis. Aust J Biol Sci 30:269-277, 1977
4. Everist SL: Poisonous Plants of Australia, pp. 465- 469. Angus and Robertson Publishers, London, 1974
5. Stalker MJ, Hayes MA: The liver and biliary system. In: Jubb, Kennedy, and Palmers Pathology of Domestic Animals, ed. Maxie MG, 5th ed., vol. 2, pp. 370-381. Elsevier Limited, St. Louis, MO, 2007

A virtual slide is not available for this case.

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