JPC SYSTEMIC PATHOLOGY
DIGESTIVE SYSTEM
October 2021
D-T03

Signalment (JPC #1851823): 1.5-year-old Holstein heifer

HISTORY: One batch of corn that was fed four to six weeks prior to this animal’s death was noticeably moldy.

HISTOPATHOLOGIC DESCRIPTION: Liver: Approximately 60% of the hepatic parenchyma is characterized by loss of normal hepatic cord architecture and replacement by abundant fibrous connective tissue (fibrosis) and increased numbers of small bile ducts lined by epithelial cells with a high nuclear to cytoplasmic ratio and loss of nuclear polarity (ductular reaction). Individual hepatocytes are multifocally dissociated and shrunken with hypereosinophilic cytoplasm and pyknotic or karyorrhectic nuclei (necrosis/single cell death). Remaining hepatocytes lack normal plate architecture, are often two to three times normal size, and have abundant eosinophilic, vacuolated cytoplasm and large vesicular nuclei with marginated chromatin and a prominent nucleolus (megalocytosis); there is moderate anisokaryosis. There are multifocal regenerative nodules that are up to 1 mm in diameter and are composed of disorganized hepatic cords that lack portal areas, are surrounded by fibrosis, and compress adjacent hepatocytes. Within these regenerative nodules, hepatocytes often contain discrete, clear, intracytoplasmic vacuoles that are up to 20 um in diameter and peripheralize the nucleus (lipid-type vacuolar change). There are scattered aggregates of neutrophils, lymphocytes, plasma cells, fewer histiocytes, and eosinophils, and rare hepatocellular necrosis/single cell death. Multifocally, portal lymphatics are mildly ectatic.

MORPHOLOGIC DIAGNOSIS: Liver: Hepatocellular necrosis and loss, chronic, diffuse, severe, with fibrosis, biliary ductular reaction, megalocytosis, and nodular regeneration with lipid-type vacuolar change, Holstein, bovine.

ETIOLOGIC DIAGNOSIS: Hepatic aflatoxicosis

CAUSE: Aflatoxin (mycotoxin B₁) elaborated by Aspergillus flavus

CONDITION: Aflatoxicosis

SYNONYM: Groundnut poisoning

GENERAL DISCUSSION:

• Aflatoxins: Bisfuranocoumarin compounds produced as metabolites by fungi such as Aspergillus flavus, Aspergillus parasiticus, and Penicillium puberlum
  • These fungi are common in the soil, air, seed, and forage worldwide; most often associated with stored or unharvested matures grains (moldy peanuts, cottonseed, soybeans, corn, rice, and wheat)
  • High humidity and temperature favor fungal growth
• Most pathogenic metabolites – B₁, B₂, G₁, and G₂
  • B and G refer to blue (B) and green (G) color reaction to fluorescent light and chromatographic values
  • Aflatoxin B₁ (AFB1) and its metabolites are the most toxic to the liver; aflatoxin M1 (a metabolite of AFB1) is found in milk and meat from cattle that have ingested AFB1
• All species of animals are susceptible; toxicity is affected by the dosage, length of exposure, animal species, and age
  • Dogs, rats, ducks, turkeys, guinea pigs, calves, pigs, horses most susceptible to toxicity
  • Sheep, adult cattle most resistant
  • Young animals are most severely affected; may die within few hours
  • Prolonged exposure to low concentrations is a more common problem than acute toxicity in large animal species
• High quality dietary protein diminishes the effects of aflatoxin
• The B6C3F1/N mouse (F1 offspring of C57BL6/N and C3H/HeN mice) is a likely animal model of human hepatocellular carcinoma; the mutations present in human and mouse hepatocellular carcinomas induced by aflatoxin B₁ are remarkably similar; Ctnnb1 gene mutations are only present in chemical induced hepatocellular carcinomas, while Hras mutations are present for both spontaneous and chemical induced hepatocellular carcinomas (Auerbach, Toxicol Pathol, 2018)

PATHOGENESIS:

• Metabolized by the P-450 dependent mixed function oxidase system in hepatic smooth endoplasmic reticulum into a variety of toxic and non-toxic metabolites
• AFB1 > metabolized to AFB-8-9 epoxide > forms a conjugate with glutathione via glutathione-s-transferase > conjugate is excreted
  • Toxicity results when glutathione-s-transferase levels are low, and unconjugated AFB-8-9 epoxide binds to macromolecules (especially nucleic acids and nucleoproteins in mitochondrial and nuclear DNA > G to T mutational transversion)
  • Differing levels of glutathione-s-transferase may cause variability in toxicity between species
• The proportions of toxic compounds produced and resulting clinical signs vary greatly with age, diet, sex, species, and environmental influences
• Megalocytes are the result of anti-mitotic effects of the toxin, which prevent cell division but not DNA synthesis because the hepatocytes attempt to divide to replace those that have undergone necrosis (inhibition of hepatocellular regeneration)
• Aflatoxins have anti-coagulative, immunosuppressive, hepatocarcinogenic, hepatotoxic, nasal mucosa pro-carcinogenic, and teratogenic effects
  • Hepatotoxicity: AFB1 oxide binds mitochondrial and nuclear DNA > inhibits biosynthetic processes and respiration, mRNA synthesis and ribosomal protein synthesis > cellular degeneration and necrosis
  • Carcinogenicity/genotoxicity: Toxins intercalate into nuclear and mitochondrial DNA > mutagenic adducts with guanosine > p53 mutation > loss of ability to prevent propagation of genetically damaged cells
  • Coagulopathy: Due to decreased hepatic synthesis of coagulation factors V, VII, VIII, and fibrinogen; not from Vitamin K antagonism (which affects factors II, VII, IX, and X); in acute aflatoxicosis with severe hepatic necrosis, disseminated intravascular coagulation (DIC) can cause coagulopathy
  • Immunosuppression: Aflatoxins suppress lymphocyte response to mitogens such as concanavalin A, phytohemagglutinin, and pokeweed mitogen, suggesting suppression of both cell-mediated and humoral immunity; lymphoid depletion occurs in the spleen, lymph nodes, bursa of Fabricius and thymus; these effects may play a role in increased susceptibility to both infections and neoplasia

TYPICAL CLINICAL FINDINGS:

• Variable; sudden death to prolonged chronic liver disease with reduction of feed efficiency, reduced daily gain, rough hair coat, enlarged abdomen, mild icterus, and eventually depression and anorexia
• Epistaxis and hemorrhagic enteritis
• Cattle: Also, blindness, photosensitive dermatitis, keratoconjunctivitis, diarrhea, severe tenesmus, abortion, and anal prolapse
• Pigs: Also, intermittent or hemorrhagic diarrhea
• Avian species: Reductions in growth, carcass pigmentation, egg production, and immune function
• Clinical pathologic changes: Anemia, increased liver enzymes (AST, ALT, ALP, GGT), total bilirubin, prothrombin time, partial thromboplastin time; decreased serum total iron-binding capacity, total protein, albumin, cholesterol, blood urea nitrogen, and glucose

TYPICAL GROSS FINDINGS:

• Liver, chronic: Pale and firm, post-necrotic fibrosis with nodular regeneration (cirrhosis), bile duct hyperplasia
• Liver, acute: Widespread hemorrhage and massive hepatic necrosis, hepatomegaly
• Ascites, mesenteric edema, widespread petechia or ecchymoses, hemorrhagic gastroenteritis; icterus
• Edema of the gallbladder is consistently seen in dogs and pigs
• Poults, pheasants, chicks, ducklings: Jaundice, edema, hemorrhages, pale/tan liver with bile duct hyperplasia; catarrhal enteritis; swelling of kidneys with severe intoxication

TYPICAL LIGHT MICROSCOPIC FINDINGS:

• Chronic (most common)
  • Bile duct proliferation
  • Megalocytosis (increase in cell and nuclear size) in the liver and kidney (proximal tubular epithelium)
  • Focal hepatocyte necrosis or apoptosis
  • Hepatic fibrosis and nodular regeneration (cirrhosis)
  • More severely affected livers: Bile pigment accumulation in canaliculi and hepatocytes
  • Fatty change variable in extent and occurrence
• Acute hepatocellular necrosis (less common)
  • Periportal: Turkeys, ducklings, chickens, adult rats, cats
  • Midzonal: Rabbits
  • Centrilobular: Pigs, cattle, dogs, guinea pigs
  • Diffuse: Neonatal rats, trout

ADDITIONAL DIAGNOSTIC TESTS:

• Tissue (liver, kidney, milk, urine) and/or analysis of contaminated feed
  • Two dimensional thin layer chromatography
  • High performance pressure liquid chromatography
  • High-performance liquid chromatography-fluorescence can quantify aflatoxin in the liver at concentrations lower than 0.2 ng/g (Shao, J Vet Diagn Invest, 2016)

DIFFERENTIAL DIAGNOSIS:

• Pyrrolizidine alkaloids (D-T04, Senecio , Crotalaria sp., Heliotropium sp.): Similar liver lesions with marked megalocytosis, +/- pulmonary lesions
• Phomopsin (Phomopsis leptostromiformis; a parasitic fungus on lupines): Diffuse mild fibrosis, numerous mitotic figures (many abnormal), bile duct proliferation, photosensitization
• Lantana (D-T12, Lantana camara): Bile retention, severe icterus, photosensitization, enlarged gallbladder, megalocytosis, cytosegrosome formation, +/- bile duct proliferation, renal nephrosis
• Copper (D-T05): Significant variation between species; hepatocellular necrosis, fibrosis, intravascular hemolysis, jaundice, hemoglobinuria, sudden death (sheep), and chronic active hepatitis (dogs)
• Sporidesmin (D-T13, Pithomyces chartarum): Facial eczema, toxic hepatitis, cirrhosis, photosensitization, pasture plants (especially dead ryegrass)
• Anticonvulsant therapy in companion animals
• Idiosyncratic drug-induced hepatotoxicity (e.g. sulfonamides in dogs): Usually more acute

COMPARATIVE PATHOLOGY:

• Aflatoxicosis has been reported in numerous domestic and laboratory animal species
• Galliformes and columbiformes: High risk of exposure due to seed-based diets; causes immunosuppression, anemia, hemorrhage, hepatic degeneration, and paralysis
• Waterfowl: Exposure suspected to arise from feeding on moldy feed left in pastures; younger animals are more susceptible, young ducks have pallor and organomegaly of liver and kidneys, with multiorgan petechiation; older ducks have firm, shrunken liver with reticular pattern, hydropericardium, and ascites
• Osteichthyes (boney fish): Exposure can be acute to chronic, the most common exposure is from contaminated feed; susceptibility to disease and neoplastic transformation is species-dependent; of increasing economic importance due to aquaculture
• Neoplasms associated with chronic aflatoxicosis:
  • Cholangiocarcinomas: Ducks 10 to 18 months of age after chronic, low- level aflatoxin feeding; hamsters also susceptible
  • Hepatocellular carcinoma: Mammals and trout
  • Hepatomas: Trout
  • Miscellaneous aflatoxin induced carcinomas: Cattle (tumors of the nasal mucosa); rats (carcinoma of the esophagus, glandular stomach, colon, and kidney)

REFERENCES:

1. Arana S, Alves VA, Sabino M, et al. Immunohistochemical evidence for myofibroblast-like cells associated with liver injury induced by aflatoxin B1 in rainbow trout (Oncorhynchus mykiss). J Comp Pathol. 2014; 150(2-3): 258-265.
2. Auerbach SS, Xu M, Merrick BA, et al. Exome sequencing of fresh frozen or formalin fixed paraffin embedded B6C3F1/N mouse hepatocellular carcinomas arising either spontaneously or due to chronic chemical exposure. Toxicol Pathol. 2018;46(6):706-718.
3. Barthold SW, Griffey SM, Percy DH, eds. Pathology of Laboratory Rodents and Rabbits, 4^th Ames, IA:John Wiley and Sons. 2016:316.
4. Boulianne M, et al. Avian Disease Manual. 8^th Jacksonville, FL: AAAP; 2019:120-125, 199.
5. Brown DL, Van Wettere AJ, Cullen JM. Hepatobiliary system and exocrine pancreas. In: McGavin MD, Zachary JF, eds. Pathologic Basis of Veterinary Disease. 6th ed. St. Louis, MO: Elsevier; 2017:450.
6. Crespo R, Franca MS, Fenton H, Shivaprasad HL. Galliformes and Columiformes. In: Terio KA, McAloose D, St. Leger J, eds., Pathology of Wildlife and Zoo Animals. San Diego, CA:Elsevier. 2018:750.
7. Constable PD, Hinchcliff KW, Done SH, Grunberg W, eds. Veterinary Medicine, A Textbook of the Diseases of Cattle, Horses, Sheep, Pigs, and Goats. 11th ed. St. Louis, MO: Elsevier; 2017:649-650.
8. Ensley SM, Radke SL. Mycotoxins in grains and feeds. In: Zimmerman JJ, Karriker LA, Ramirez A, Schwartz KJ, Stevenson GW, Zhang J, eds. Diseases of Swine. 11th ed. Hoboken, NJ: John Wiley & Sons, Inc.; 2019:1055-1060.
9. Fenton H, McManamon R, Howerth EW. Anseriformes, Ciconiiformes, Charadriiformes, and Gruiformes. In: Terio KA, McAloose D, St. Leger J, eds., Pathology of Wildlife and Zoo Animals. San Diego, CA:Elsevier. 2018:701-707, 717.e2.
10. Frasca S, Wolf JC, Kinsel MJ, Camus AC, Lombardini ED. Osteichthyes. In: Terio KA, McAloose D, St. Leger J, eds., Pathology of Wildlife and Zoo Animals. San Diego, CA:Elsevier. 2018:957.
11. Hoerr FJ. Mycotoxicoses. In: Swayne DE, ed. Diseases of Poultry. 14th ed. Hoboken, NJ: John Wiley & Sons, Inc.; 2020:1330-1339.
12. Qian G, Wang F, Tang L. Integrative toxicopathological evaluation of aflatoxin B₁ exposure in F344 rats. Toxicol Pathol. 2013; 41(8): 1093-1105.
13. Shao D, Imerman PM, Schrunk DE, Ensley SM, Rumbeiha WK. Intralaboratory development and evaluation of a high-performance liquid chromatography-fluorescence method for detection and quantitation of aflatoxins M1, B1, B2, G1, and G2 in animal liver. J Vet Diagn Invest. 2016;28(6):646-655.
14. Uzal FA, Plattner BL, Hostetter JM. Alimentary system. In: Maxie MG, ed. Jubb, Kennedy, and Palmer’s Pathology of Domestic Animals. Vol 2. 6th ed. St. Louis, MO: Elsevier; 2016:333-334.
15. Wouters ATB, Casagrande RA, Wouters F, et al. An outbreak of aflatoxin poisoning associated with aflatoxin B1-contaminated maize products. J Vet Diagn Invest. 2013; 25(2): 282-287.

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