JPC SYSTEMIC PATHOLOGY

DIGESTIVE SYSTEM

October 2024

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 µm 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 (most important sources of aflatoxins)
  • 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
    • AFB1 is a naturally occurring carcinogen and therefore has important public health concerns 
• 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
    • Acute conditions in horses and cattle are rare due to the large quantity needed to cause an effect
• High quality dietary protein diminishes the effects of aflatoxin

 

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
  1. Metabolites bind to RNA and DNA and can alter cell division = carcinogenicity due to DNA adduct formation
  2. Binding to DNA can also result in disruption of pathways of protein synthesis causing issues in metabolic pathways like the Kreb’s cycle, the clotting cascade, lipid metabolism and the host immune system (due to altered globulin synthesis)
• 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 – indicating that toxicity genetically determined by its biosynthesis to its metabolites
  • AFB-8-9 epioxide metabolite is the most potent
• Aflatoxin has both cytotoxic and mutagenic properties = direct hepatocellular damage and neoplastic transformation
• 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, nephrotoxic, 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 (ill-thrift animals)
  1. Chronic toxicosis is much more common in all species
  2. Targets liver, kidney, immunity, hematopoiesis, reproduction, digestion, integument and the musculoskeletal system
• 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, decrease egg production, and immune function
• Rabbits: Coagulation defects with diminished synthesis of coagulation factors
  1. In severe cases of hepatic necrosis, intravascular coagulation and consumption of coagulation factors takes place
• Free range birds: Blindness, unresponsiveness, depression, tremors, inability to fly
• 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 
• Aflatoxin influences calcium and phosphorus metabolism by altering the metabolism of vitamin D and parathyroid hormone
• It also effects intestinal crypt epithelium altering digestion 
• Dose rate of intoxication is as little as < 1.0mg/kg
• In chickens: depresses complement activity, decreases phagocytic activity, impairs cell-mediated immunity through inhibition of thymic-associated lymphocytes in chickens

 

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 
  1. Shrunken and fibrotic liver from collapse of necrotic areas and condensation of the fibrous stroma
• 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 
• Chickens: Yellow to ocher discoloration of the liver with a reticulated pattern on the capsule and white foci 

 

TYPICAL LIGHT MICROSCOPIC FINDINGS:

• Chronic (most common)
  • Bile duct proliferation (biliary hyperplasia)
  • Megalocytosis (increase in cell and nuclear size) in the liver and kidney (proximal tubular epithelium) 
    • Prominent nucleoli in hepatocytes 
  • Focal hepatocyte necrosis or apoptosis
  • Hepatic fibrosis and nodular regeneration (cirrhosis) – as the liver becomes atrophic
  • More severely affected livers: Bile pigment accumulation in canaliculi and hepatocytes
  • Fatty change variable in extent and occurrence (steatosis of hepatocytes)
  • Periportal necrosis and inflammation (due to toxin being excreted in the bile)
  • Turkeys: nodules of regenerative hepatocytes 
• Acute hepatocellular necrosis (less common)
  • Periportal: Turkeys, ducklings, chickens, adult rats, cats
  • Midzonal: Rabbits, horses, pigs
  • Centrilobular: Pigs, cattle, dogs, guinea pigs
  • Diffuse: Neonatal rats, trout
  • Within the kidneys – degeneration of the proximal convoluted tubules; thickening of glomerular basement membranes (particularly in birds)
• The microscopic changes to the liver are expected due to toxic insult to this organ and are not pathognomonic for aflatoxin
• At higher doses, aflatoxin causes centrilobular hepatocyte loss with replacement by mixed inflammation, fibroblasts, and primitive vascular channels.

ADDITIONAL DIAGNOSTIC TESTS: 

• Sample of choice for testing is feed
  1. Using ELISA and radioimmunoassays
  2. Freezing feed samples is recommended if there is a delay in testing
  3. Gastrointestinal content may be used
  4. It is extremely difficult to detect mycotoxins in tissue samples due to rapid degradation of the toxin
     • High performance pressure liquid chromatography needed
  5. Some labs can test for metabolites but this is not very sensitive
  6. Cases of mycotoxins are often suspected but not confirmed
• In fish, liver can be used for testing based on bioaccumulation 

DIFFERENTIAL DIAGNOSIS:

• Pyrrolizidine alkaloids (D-T04, Senecio sp., 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
• Cockatiels are very sensitive to this toxin
• 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
• Rabbits are among the most sensitive to aflatoxin B1
• Fish are also considered highly sensitive 
• 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:

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