JPC SYSTEMIC PATHOLOGY
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 chord architecture with hepatocytes being replaced 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 shrunken, dissociated and hypereosinophilic with pyknotic or karryrhectic nuclei (necrosis). Remaining hepatocytes are disorganized and 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 aniokaryosis. There are multifocal regenerative nodules which measure up to 1mm in diameter, lack portal areas, compress adjacent hepatocytes and are composed of disorganized hepatic cords surrounded by fibrosis. Regenerative hepatocytes often contain 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 single hepatocellular necrosis. Multifocally, portal lymphatics are mildly ectatic (edema).
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
SYNONYM: Groundnut poisoning
- Aflatoxins: Bisfuranocoumarin compounds produced as metabolites by fungal agents such as Aspergillus flavus, Aspergillus parasiticus, and Penicillium puberlum
- 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 a more common problem than acute toxicity in large animal species
- High quality dietary protein diminishes the effects of aflatoxin
- 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 procarcinogenic, 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
- Pyrrolizidine alkaloids (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 (Lantana camara): Bile retention, severe icterus, photosensitization, enlarged gallbladder, megalocytosis, cytosegrosome formation, +/- bile duct proliferation, renal nephrosis
- Copper: Significant variation between species; hepatocellular necrosis, fibrosis, intravascular hemolysis, jaundice, hemoglobinuria, sudden death (sheep), and chronic active hepatitis (dogs)
- Sporidesmin (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
- Aflatoxicosis has been reported in numerous domestic and laboratory animal species
- 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); rabbits and mice (lung tumorigenesis)
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