JPC SYSTEMIC PATHOLOGY

URINARY SYSTEM

November 2023

U-B06

 

Signalment (JPC #1545652): A lamb

 

HISTORY: A rapidly growing lamb found dead in its pasture. Petechiae were noted on most serosal surfaces and fluid was found in the pericardial sac.

 

HISTOPATHOLOGIC DESCRIPTION: Kidney: Affecting approximately 90% of the cortical tubules, there is coagulative necrosis characterized by retention of cellular architecture, poorly defined epithelial cell borders, loss of differential staining, and loss of nuclear detail or fragmentation of nuclei (karyolysis). Tubular epithelial cells are often detached from the intact basement membranes. Few remaining tubules within the cortex, and tubules within the medulla, are lined by necrotic epithelial cells with hypereosinophilic cytoplasm and nuclear pyknosis, karyorrhexis, or karyolysis.  Multifocally, glomeruli contain small foci of eosinophilic cellular and karyorrhectic debris (lytic necrosis) admixed with fibrin and/or are segmentally to globally congested.  Multifocally within the interstitium in the cortex and medulla, there is mild hemorrhage, fibrin, edema, and congested blood vessels. Occasionally, medullary tubules contain small amounts of amorphous intensely basophilic mineral. There is undulation of the capsular connective tissue. Interstitial blood vessels often have thickened walls due to edema and fibrin accumulation, there is degeneration and/or necrosis of endothelium and vascular smooth muscle, and perivascular edema and hemorrhage. 

 

MORPHOLOGIC DIAGNOSIS: Kidney, tubules: Necrosis, coagulative, diffuse, with multifocal mild interstitial hemorrhage, breed unspecified, ovine.

 

ETIOLOGIC DIAGNOSIS: Enterotoxemic nephrosis

 

CAUSE: Clostridium perfringens Type D

 

SYNONYMS: Enterotoxemia; pulpy kidney disease; overeating disease; braxy-like disease.

 

GENERAL DISCUSSION: 

• “Pulpy kidney disease” is historically considered the renal manifestation of Clostridium perfringens Type D enterotoxemia in sheep and goat; however renal changes may be due to nonspecific postmortem changes/advanced autolysis 
  1. Recent studies have found no specific gross or histologic renal lesions in sheep with experimental acute type D enterotoxemia, including no more rapid autolysis than other causes; therefore “pulpy kidney disease” can be misleading and should be avoided (Giannitti, Vet Pathol. 2023)
• C. perfringens is an anaerobic, spore-forming, gram positive bacillus, found worldwide in soil, manure, and as an intestinal commensal in ruminants  

• C. perfringens is classified into 5 major types (A, B, C, D, and E), based on the production of 4 major lethal toxins:
  • C. perfringens Type D produces alpha toxin and epsilon toxin
    • Epsilon toxin (ETX) is the third most potent clostridial toxin (after botulinum and tetanus toxins)
    • Some isolates produce up to five different toxins (may also play a role in virulence), although ETX is necessary to induce disease
    • See chart below for more information

• C. perfringens Type D enterotoxemia is an acute, infectious, noncontagious disease of sheep and goats, and occasionally cattle, and is characterized by convulsions, hyperglycemia, and sudden death
  1. In sheep, it rarely causes Focal Symmetrical Encephalomalacia (FSE) (N-B01), most commonly in lambs >2 weeks or adult sheep
  2. In goats, it causes enterocolitis and/or neurological disease
  3. All ages except newborns are susceptible
     • Newborns lack pancreatic proteolytic enzymes (trypsin and trypsin-chymase) necessary for activation of epsilon toxin, due to trypsin inhibitors in colostrum which prevent breakdown of immunoglobulins during passive transfer

 

PATHOGENESIS: 

• Clostridial spores ingested from environment (can be normal inhabitant); Alterations in sheep intestinal environment due to sudden diet changes (e.g., sudden high starch diets (lush spring pastures or high grain diet)) à Undigested starch in the small intestine à Increased bacterial substrate and subsequent C. perfringens type D overgrowth à Low environmental glucose leads to epsilon exotoxin production à Epsilon toxin activated by cleavage by trypsin and/or chymotrypsin à Epsilon toxin facilitates its own absorption through the intestinal mucosa à Endothelial damage and increased vascular permeability (especially in lungs and brain)
  1. Kidney lesions: Epsilon toxin acts directly on tubular epithelial cell membranes (tubular epithelium expresses epsilon toxin receptors) as well as on renal endothelium 
     • Endothelial damage à Acute coagulative necrosis 
     • Tubular epithelium damage à Membrane-mediated cytotoxicity and cell lysis
  2. Brain lesions: Endothelial damage and blood-brain-barrier breakdown à Increased vascular permeability à Vasogenic brain edema à Hypoxic-ischemic necrosis à Death

• Gene for epsilon toxin (and many other clostridial toxins) are located on plasmids
• ETX/epsilon toxin:

1. An angiotoxic permease that alters cell permeability (pore-forming toxin)
   • Alpha toxin is a phospholipase toxin

• ETX binds to endothelial cells, resulting in:
  • Opening of tight junctions
  • Disturbed transport processes
  • Increased vascular permeability
  • Swelling of astrocytic foot processes
  • Necrosis due hypoxic-ishemic mechanisms
• Some effects may be mediated by the adenyl cyclase/cAMP system 
• ETX is endotheliotoxic and may damage the blood-brain-barrier (Mander, J Vet Diagn Invest 2020)
• Causes microvascular endothelial injury in the retina leading to vasogenic edema; large doses of toxin may cause visual deficits (Finnie, J Vet Diagn Invest 2020)
• May lead to cardiopulmonary lesions (edema, cardiac hemorrhage) (Giannitti, Vet Pathol. 2021)
• May be activated by lambda toxin, a clostridial protease that activates ETX in vitro; this resulted in fatal diarrhea in 2 goat kids younger than 2 weeks of age (Acevedo, J Vet Diagn Invest. 2023)

 

TYPICAL CLINICAL FINDINGS: 

• Clostridium perfringens Type D enterotoxemia affects fattening sheep, goats, and calves and is associated with grain overload (“overeating disease”)
• Sheep:
  • Acute: 3-10-week-old, fast-growing lambs on a high nutritional plane; acute neurological and respiratory signs (convulsions and tachypnea, bawling as from severe pain, sudden death)  
  • Subacute or chronic: Focal symmetric encephalomalacia with neurologic signs (blindness, ataxia, head pressing, seizures) and death; occasionally diarrhea; affects older sheep
• Goats: 
  • Similar to sheep, more frequently seen in adult goat; subacute disease with hemorrhagic diarrhea most common; Chronic: Profuse, watery diarrhea (often containing blood and mucus)

 

TYPICAL GROSS FINDINGS:

• “Pulpy kidney” (softening of the renal parenchyma): Congested, soft kidneys, best regarded as rapid postmortem autolysis; inconsistent and of little diagnostic value
• Multifocal hemorrhage and edema
  1. Pulmonary congestion and edema, also hydrothorax; ascites; hydropericardium; endocardial, myocardial and epicardial hemorrhages 
  2. Serosal petechiation and hemorrhage also on thymus, GI tract, renal cortex 
• Brain: Focal and bilaterally symmetric encephalomalacia in lambs and older sheep most often affecting the corpus striatum, thalamus, cerebellar peduncles most common with white matter is preferentially affected; cerebellar coning (herniation of the cerebellar vermis)

• Goats: Usually severe necrotizing colitis and/or enterocolitis +/- mesocolonic edema, hyperemia, and mucosal ulceration 

 

TYPICAL LIGHT MICROSCOPIC FINDINGS:

• Kidney: Consistently histologic changes are not observed, either in “pulpy kidneys” or kidneys experimentally inoculated (supporting that gross changes are postmortem autolysis)
• Sheep
  1. Subendocardial, subserosal, or subperitoneal hemorrhage
  2. Brain: 
     • Acute and subacute: Perivascular proteinaceous edema (microangiopathy): homogeneous acidophilic accumulations of protein surrounding small- and medium-sized arteries and veins; with hypertrophied vascular endothelium and hyalinization of arteriolar walls
       • Unique and pathognomonic; observed in ∼90% of cases
     • Neuropil interstitial (vasogenic) edema with a light-pink, spongy appearance to parenchyma; pericapillary hemorrhage, acute neuronal and microglial necrosis; white matter degeneration, and astrocyte and axonal swelling with spheroid formation
     • With longer survival, there may be axonal swelling, accumulation of neutrophils and foamy macrophages present
     • FSE (subacute and chronic) has bilaterally symmetrical perivascular proteinaceous edema

• Heart: Acute myocardial degeneration and necrosis; myocardial, endocardial, and epicardial edema, hemorrhage, and inflammation (Giannitti, Vet Pathol. 2021)

• Goats: 
  1. Subacute and chronic: Suppurative and fibrinonecrotizing enterocolitis most commonly in the colon and caudal small intestine
  2. Perivascular edema occasionally observed; can rarely see FSE-type lesions in the brain
     • Recent report demonstrated that 18% of goats with C. perfringens type D enterotoxemia had microscopic brain lesions, including previously undescribed intramural proteinaceous vascular edema (rather than perivascular as described in sheep); pulmonary congestion and edema, hydrothorax, hydropericardium, and ascites were also described (Ortega, Vet Pathol. 2019) 

 

ULTRASTRUCTURAL FINDINGS:

 

ADDITIONAL DIAGNOSTIC TESTS:

• Demonstration of ETX/epsilon toxin in the blood or ileal contents
• A panel of antibodies (amyloid precursor protein, 3 subunits of neurofilament protein, protein gene product 9.5, ubiquitin, and synaptophysin) can be used to detect axonal swellings or spheroid in FSE (Manavis, Vet Pathol. 2022).

 

DIFFERENTIAL DIAGNOSIS:

For sudden death in lambs:

• Grain overload
• Yellow lamb disease (C. perfringens Type A)
• Lamb dysentery (C. perfringens Type B)
• Hydrocyanic acid plant associated toxicosis (cyanide poisoning - chokecherry, arrowgrass, Johnson grass, etc)
• Braxy (Clostridium septicum) 
• Black disease (Clostridium novyi)

 

COMPARATIVE PATHOLOGY: 

• C. perfringens associated with acute gastric dilatation in NHPs 
• Cattle: Natural cases of enterotoxemia rare; proteinaceous perivascular edema of the brain and lungs in calves.

• Camelids: C. perfringens type D enterotoxemia suspected in camelids with serosal hemorrhages, pericardial fibrin clots, and pulmonary and cerebral edema 
• C. perfringens also causes clostridial myositis; a necrotizing infection of subcutaneous tissue and muscle; affects mainly ruminants and horses
  1. In cattle, more commonly linked to nonpenetrating trauma -> muscle bruising and anaerobic conditions that activate clostridial spores already in the muscle
  2. In horses is usually secondary to a penetrating wound
  3. Clostridial myositis may also be caused by Clostridium chauvoei, C. septicum, C. novyi type A, C. perfringens type A, and C. sordellii 
     • C. perfringens often causes gas gangrene while malignant edema is often due to C. septicum
     • May be linked with C. perfringens types A-E (Valentine, 2022) or specifically C. perfringens types A (Junior, J Vet Diagn Invest. 2020). 
• Diseases caused by C. perfringens:

 

Clostridium perfringens – Types, toxins, and diseases
Type	Toxin						Diseases
	Alpha	Beta	Epsilon	Iota	CPE	NetB
A	+	-	-	-	-	-	Gas gangrene Yellow lamb disease – enterotoxemia (western US) Colitis X – horses Hemorrhagic bowel syndrome – dairy cattle Food Borne Illness - humans Necrotic enteritis - chickens Gastroenteritis - ferrets  Necrotizing enterocolitis-piglets Enterotoxemia-calves and lambs Canine hemorrhagic gastroenteritis
B	+	+	+	-	-	-	Lamb dysentery Hemorrhagic enteritis – calves, foals, GPs (UK, S. Africa, Middle East) Hemorrhagic enterotoxemia-sheep
C	+	+	-	-	+/-	-	Enterotoxic hemorrhagic enteritis - neonatal lambs, goats, cattle, pigs Struck – adult sheep, hemorrhagic enteritis & peritonitis (UK)
D	+	-	+	-	+/-	-	Overeating disease/pulpy kidney - Sheep, cattle, goats Enterocolitis-goats Focal symmetric encephalomalacia – Sheep, goats
E	+	-	-	+	+/-	-	Enterotoxemia - calves, lambs. guinea pigs, rabbits Enteritis-lagomorphs
F	+	-	-	-	+	-	Food borne illness - humans
G	+	-	-	-	-	+	Necrotic enteritis in chickens
Additional toxins exist (such as NetE, NetF, NetG), but require further classification (Gohari, J Vet Diagn Invest. 2020). Table compiled from: Uzal, 2016, Pg 183; Uzal, Open Toxinology J. 2010; Rood, Anaerobe 2018  Additions from: Profeta. J Vet Diagn Invest. 2020

 

REFERENCES:

1. Acevedo HD, Schlesinger MS, et al. Enterotoxemia produced by lambda toxin-positive Clostridium perfringens type D in 2 neonatal goat kids. J Vet Diagn Invest. 2023;35(4):448-451.
2. Agnew D. Camelidae. In: Terio K, McAloose D, Leger J, eds. Pathology of Wildlife and Zoo Animals, San Diego, CA: Elsevier 2018: 198.
3. Cianciolo RE, Mohr FC. Urinary System. In: Maxie MG, ed. Jubb, Kennedy & Palmer's Pathology of Domestic Animals. Vol 2. 6th ed. St. Louis, MO: Elsevier; 2016:382.
4. Cooper BJ, Valentine BA. Muscle and Tendon. In: Maxie MG, ed. Jubb, Kennedy & Palmer's Pathology of Domestic Animals. Vol 1. 6th ed. St. Louis, MO: Elsevier; 2016:230-232. 
5. Finnie JW, Navarro MA, Uzal FA. Pathogenesis and diagnostic features of brain and ophthalmic damage produced by Clostridium perfringens type D epsilon toxin. J Vet Diagn Invest. 2020;32(2):282-286.
6. Giannitti F, García JP, et al. Cardiopulmonary Lesions in Sheep Produced by Experimental Acute Clostridium Perfringens Type D Enterotoxemia. Vet Pathol. 2021;58(1):103-113.
7. Gohari IM, Unterer S, Whitehead AE, Prescott JF. NetF-producing Clostridium perfringens and its associated diseases in dogs and foals. J Vet Diagn Invest. 2020;32(2):230-238.
8. Junior CAO, Silva ROS, et al. Gas gangrene in mammals: a review. J Vet Diagn Invest. 2020;32(2):175-183. 
9. Manavis J, Blumbergs P, Jerrett I, Hanshaw D, Uzal F, Finnie J. Heterogeneous immunoreactivity of axonal spheroids in focal symmetrical encephalomalacia produced by Clostridium perfringens type D epsilon toxin in sheep. Vet Pathol. 2022;59(2):328-332.
10. Mander KA, Uzal FA, Williams R, Finnie JW. Clostridium perfringens type D epsilon toxin produces a rapid and dose-dependent cytotoxic effect on cerebral microvascular endothelial cells in vitro. J Vet Diagn Invest. 2020;32(2):277-281.
11. Matz-Rensing K, Lowenstine LJ. New World and Old World Monkeys. In: Terio K, McAloose D, Leger J, eds. Pathology of Wildlife and Zoo Animals, San Diego, CA: Elsevier 2018: 363.
12. Miller AD, Porter, BF. Nervous System. In: Zachary JF, ed. Pathologic Basis of Veterinary Disease. 7th ed. St. Louis, MO: Elsevier; 2022:974-975.
13. Ortega J, Verdes JM, et al. Intramural vascular edema in the brain of goats with Clostridium perfringens type D enterotoxemia. Vet Pathol. 2019;56(3):452-459.
14. Profeta F, Di Francesco CE, et al. Prevalence of netB-positive Clostridium perfringens in Italian poultry flocks by environmental sampling. J Vet Diagn Invest. 2020;32(2):252-258.
15. Rood JI, Adams V, Lacey J, et al. Expansion of the Clostridium perfringens toxin-based typing scheme. Anaerobe. 2018;53:5-10.
16. Spagnoli ST, Gelberg HB. Alimentary System and the Peritoneum, Omentum, Mesentery, and Peritoneal Cavity. In: Zachary JF, ed. Pathologic Basis of Veterinary Disease. 7th ed. St. Louis, MO: Elsevier; 2022:451-452, 472.
17. Simmons J, Gibson S. Bacterial and mycotic diseases of nonhuman primates. In: Abee CR, Mansfield K, Tardif S, Morris T. Nonhuman Primates in Biomedical Research Volume 2: Diseases. 2nd ed. London, UK: Academic Press; 2012:122-123.
18. Stanton JB, Zachary JF. Mechanisms of Microbial Infections. In: Zachary JF, ed. Pathologic Basis of Veterinary Disease. 7th ed. St. Louis, MO: Elsevier; 2022:203-204, 226. 
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20. 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. Philadelphia, PA: Elsevier Ltd; 2016: 183-191.  
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