JPC SYSTEMIC PATHOLOGY
DIGESTIVE SYSTEM
October 2021
D-T04

SLIDE A: Signalment (JPC #2548697):  An 8-month-old Holstein steer

HISTORY:  This animal died while being transported to a diagnostic laboratory.  The steer had suddenly stopped eating and became lethargic the day before.  The only significant gross lesion was a pale and extremely firm liver.  Groundsel (Senecio spp.) was identified in the hay that had been fed to this animal.

HISTOPATHOLOGIC DESCRIPTION:  Liver:  Diffusely, there is loss of hepatic cord architecture characterized by dissociation of hepatic cords and multifocal loss of hepatocytes with replacement by intersecting bands of variably mature fibrous connective tissue that expand and bridge centrilobular and portal zones (bridging fibrosis), extend into and replace approximately 50% of the normal hepatic cord architecture, and separate, surround, and individualize hepatocytes.  The fibrous bands are admixed with markedly increased numbers of biliary duct profiles (ductular reaction).  Multifocally, centrilobular fibrosis surrounds, compresses, and occasionally obliterates central veins (veno-occlusion).  Multifocally, hepatocytes are characterized by one of the following: mildly swollen with pale, vacuolated cytoplasm (degeneration), less frequently have abundant cytoplasm and nuclei up to twice normal size (megalocytosis) with rare intranuclear cytoplasmic invaginations, and occasionally are shrunken with hypereosinophilic cytoplasm and pyknotic or karryorhectic nuclei (single cell death).

MORPHOLOGIC DIAGNOSIS:  Liver: Hepatocellular degeneration and loss, diffuse, severe, with centrilobular and portal bridging fibrosis, marked biliary ductular reaction, hepatic veno-occlusion, and megalocytosis, Holstein, bovine.

SLIDE B: Signalment (JPC #4049053): 9-month-old, male, sport horse

HISTORY:  Presented with a 6-week history of lesions involving the dorsal cervical and nasal skin.

HISTOPATHOLOGIC DESCRIPTION: Liver:  Diffusely, periportal and to a lesser extent midzonal sinusoids are expanded and often effaced by a florid proliferation of bile ducts (biliary ductular reaction) admixed with a moderate amount of mature collagen, hypertrophied fibroblasts (fibrosis), macrophages with intracytoplasmic brown pigment (hemosiderin, lipofuscin, or bile), and few neutrophils and lymphocytes.  In these affected areas, there is loss of normal hepatocellular architecture; hepatocytes are separated, individualized, surrounded, and often replaced by proliferating bile ducts and fibrosis.  Remaining hepatocytes are often enlarged up to 2-3 times normal (megalocytosis), with cytoplasm ranging from a lacy, pink, ground glass appearance (glycogen-type vacuolar change) to discretely microvacuolated (lipid-type vacuolar change); both changes are sometimes present within the same hepatocyte.  Many hepatocytes contain nuclei that are three times the size of surrounding hepatocytes (karyomegaly) and have open chromatin and a prominent nucleolus; low numbers of nuclei contain entrapped, eosinophilic, intranuclear cytoplasmic invaginations.  Entrapped hepatocytes are often shrunken (atrophic) and rarely are hypereosinophilic with pyknotic nuclei (single cell death), occasionally surrounded by low numbers of neutrophils.  Some hepatocytes contain abundant cytoplasmic granular brown pigment (hemosiderin or lipofuscin), and scattered macrophages within areas of biliary ductular reaction contain similar pigment.  There are scattered, up to 2mm diameter, nodules of hepatocyte hyperplasia predominantly lacking normal lobular architecture (nodular regeneration) comprising 30% of the section, and the overlying capsule is undulant.  Portal areas contain small numbers of lymphocytes, plasma cells, and rare hemosiderin-laden macrophages.  Portal lymphatics are moderately ectatic.

MORPHOLOGIC DIAGNOSIS:  Liver: Hepatocyte degeneration, necrosis, and loss, chronic, diffuse, severe, with severe biliary ductular reactoin, portal and bridging fibrosis, nodular regeneration, and hepatocellular megalocytosis, sport horse, equine.

ETIOLOGIC DIAGNOSIS:  Pyrrolizidine alkaloid hepatopathy

CAUSE:  Pyrrolizidine alkaloids

GENERAL DISCUSSION:

• Pyrrolizidine alkaloids (PA) have been identified in nearly 3% of all plant species, in >6,000 species of 3 plant families: Asteraceae, Leguminosae, and Boraginaceae; the main genera responsible for plant toxicosis in domestic mammals are Senecio, Crotalaria, Heliotropium, Cynoglossum, Amsinckia, Echium, and Trichodesma
• Intoxication is relatively infrequent because these plants are unpalatable; ingestion frequently occurs when baled or cubed forage are contaminated
• >300 pyrrolizidine alkaloids have been identified, and plants often contain more than one, but only a few have well-characterized toxicity, all of which are esters of 1 of 3 amino alcohol bases (necines) or acids (necic acids):
  • Retronecine group: monocrotaline, retrorsine, retronecine, ridelliine, senecionine, jacobine
  • Heliotridine group: lasiocarpine, heliotrine
• Most pyrrolizidine alkaloids are hepatotoxic because they are metabolically activated to dehydropyrrolizidine (DHP) alkaloids in the liver
• Young animals are generally more susceptible than adults
• Ruminants, especially sheep and goats, are much less susceptible than pigs (the toxin can be partially degraded in the rumen); horses and cattle have intermediate susceptibilities
• If seasonal exposure declines, a remarkable degree of recovery is possible

PATHOGENESIS:

• Only a small proportion of the known alkaloids have well-characterized toxicity
• Toxins are metabolized to more toxic forms by Cytochrome P450 (CYP450), which is of highest concentration in centrilobular hepatocytes (main site of pyrrolizidine alkaloid biotransformation), but also found in proximal convoluted renal tubules and club cells of the lungs (metabolites generated in both locations results in local injury) -> in some species and some cases, death can result from renal damage or pulmonary vascular and interstitial lesions
• Metabolism by CYP450 into pyrrolic esters -> N-oxidation of the necine bases -> 2-step hydroxylation of necine bases at C3 and C8 positions -> spontaneous dehydration to the highly reactive dehydropyrrolizidine (DHP) alkaloids à DHP (electrophile) binds covalently to amino acids, proteins, and nucleic acids at guanine and adenine residues ->
  • Acute toxicity: Damage to cellular proteins
  • Antimitotic effects: Damage to microtubules
  • Genotoxicity and carcinogenesis: DNA binding à inhibits DNA synthesis and mitosis in hepatocytes, some hepatocytes are able to replicate DNA but are unable to undergo mitosis à greatly enlarged hepatocytes with large, convoluted, polyploid nuclei (megalocytosis)
• There is evidence that pyrrolizidine alkaloid metabolites are toxic to endothelium as well as hepatocytes
• Detoxification of DHP: glutathione conjugation by glutathione S-transferases, or hydrolysis of ester linkages at C7 and C9 by carboxylesterases to generate necine or acid moieties
• The toxicity of pyrrolizidine alkaloid-containing plants depends on many factors:
• Toxin content (amount): Related to stage of plant growth (new growth tends to have more toxin), time of year, rainfall
  • Toxin type (which specific alkaloids): Alkaloids from Crotalaria affect the widest range of tissues in most animals, e.g. monocrotaline causes diffuse lung injury à pulmonary edema à eventual fibrosis
  • Rate of CYP450-mediated generation of DHP
  • Rate of DHP detoxification by glutathione conjugation
  • Species, age, and sex of animal exposed
• Hepatic fibrosis can result in portal hypertension with ascites, severe mesenteric edema, and diarrhea

CLINICAL FINDINGS:

• 3 disease forms:
  • Acute: Rare; due to ingestion of large amounts of these alkaloids; naturally occurring outbreaks are rare due to unpalatability of these plants
  • Phasic (usually seasonal): Repetitive exposure of these alkaloids; this is the most common form
  • Prolonged exposure to Heliotripium (exclusively): Firm, fibrotic liver without nodular regeneration
• Hepatic failure (with chronic disease): metabolic disturbances, bleeding tendencies, hypoalbuminemia, portal hypertension +/- acquired portosystemic shunt, ascites (uncommon in horses with liver failure), hepatocutaneous syndrome, photosensitization; horses are prone to hepatic encephalopathy e.g. head pressing and compulsive walking (hence colloquial names “walkabout” and “walking disease”)

TYPICAL GROSS FINDINGS:

• Livers are shrunken, fibrotic, nodular and discolored; accentuation of lobular pattern (except prolonged exposure to Heliotropium does not result in nodular regeneration)
• Ascites
• Occasionally, jaundice and photosensitization can occur

TYPICAL LIGHT MICROSCOPIC FINDINGS:

• There are 3 common morphologic expressions:
  • Acute: Centrilobular to massive necrosis +/- hemorrhage +/- fibrosis that can develop in a “veno-occlusive” pattern around and obliterating hepatic venules
  • Phasic (MOST COMMON): Characteristic megalocytosis (not pathognomonic), hepatic fibrosis, biliary proliferation, +/- nodular regeneration; hepatic atrophy; +/- prominent cytoplasmic invaginations entrapped within hepatocyte nuclei (appearing as intranuclear inclusions)
  • Chronic: Atrophic hepatocytes, biliary ductular reaction (proliferation of bile duct epithelium) in portal triads, +/- variable periportal fibroplasia (minimal in sheep, moderate in horses, marked in cattle including lobular dissecting fibrosis)
    • Prolonged exposure to Heliotropium specifically: fibrotic, atrophic livers without nodular regeneration
• Pulmonary lesions: diffuse fibrosis of alveolar and interlobular septa with patchy epithelialization

ADDITIONAL DIAGNOSTIC TESTS:

• Identification of the toxic plants in feed
• Detection of pyrrolic esters by chromatographic methods in tissue

DIFFERENTIAL DIAGNOSIS:

Hepatocellular megalocytosis in cattle:

• Nitrosamines: Megalocytosis is not as prominent and centrilobular fibrosis is less severe than pyrrolizidine alkaloids
• Aflatoxins (D-T03): Biliary hyperplasia and hemorrhagic necrosis; megalocytosis is less prominent
• Triterpenes (Lantana, D-T12): Focal necrosis, and canalicular cholestasis; icterus and photosensitization common
• Methylazoxymethanol (Cycadales): Centrilobular necrosis and cholestasis +/- CNS lesions

Causes of end-stage liver:

• Chronic toxicity – therapeutic agents or toxins
• Chronic cholangitis and/or obstruction
• Chronic congestion (right sided heart failure)
• Chronic hepatitis
• Idiopathic

COMPARATIVE PATHOLOGY:

• Alkaloids from Crotalaria affect the widest range of tissues in most animals:
  • Monocrotaline causes diffuse lung injury, leading to pulmonary edema progressing to fibrosis
• Sheep: Protracted disease course due to relative resistance; Heliotropium europaeum and Echium plantagineum are most commonly implicated; affected livers are small, gray-yellow, firm, smooth; if liver copper content was high prior to toxic insult, release of copper may lead to intravascular hemolysis à marked jaundice, gun-metal colored kidneys (i.e. copper toxicosis); develop pulmonary signs after ingesting Crotalaria (C. globifera and C. dura)
• Horses: Susceptible to acute and chronic toxicosis; liver failure is similar to cattle, although horses are more likely to manifest with hepatic encephalopathy; horses tend to have less nodular regeneration than cattle; horses can develop respiratory difficulty after ingestion of Crotalaria (C. dura and C. crispate) with marked emphysema
• Pigs: Develop pulmonary signs and emphysema after ingesting Senecio jacobaea
• Rats: Develop progressive pulmonary disease, pulmonary hypertension, and cor pulmonale with necrotizing vasculitis of pulmonary arterioles following experimental feeding of Crotalaria spectabilis or injection with monocrotaline
• Free-ranging African antelope: Senecio, Heliotropium spp. and Crotolaria spp.
  • Acute: Submassive/massive hepatic necrosis
  • Chronic: Chronic-active or piecemeal hepatitis with hepatocyte megalocytosis portal fibrosis, terminal cirrhosis

REFERENCES:

1. Brown DL, Van Wettere AJV, Cullen JM. Hepatobiliary system and exocrine pancreas. In: Zachary JF, ed. Pathologic Basis of Veterinary Disease. 6th ed. St. Louis, MO: Elsevier; 2017:429-430, 440, 440.e1, 449-450.
2. Cullen JM, Stalker MJ. Liver and Biliary system. In: Maxie MG, ed. Jubb, Kennedy and Palmer’s Pathology of Domestic Animals. Vol 2. 6th ed. St. Louis, MO: Elsevier Ltd; 2016:336-338, 519-520.
3. Jones M.E.B, Gasper DJ, Mitchell E. Bovidae, Antilocapridae, Giraffidae, Tragulidae, Hippopotamidae. In: Terio KA, McAloose D, Judy St. Leger J, eds. Pathology of Wildlife and Zoo Animals. Cambridge, MA Academic Press; 2018:141.e6.

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