JPC SYSTEMIC PATHOLOGY
DIGESTIVE SYSTEM
August 2021
D-B02

Signalment (JPC #1946317):  4-day-old Thoroughbred foal

HISTORY:  The foal presented with bloody diarrhea (frank blood) 12 hours prior to death

HISTOPATHOLOGIC DESCRIPTION:  Small intestine:  There is diffuse, circumferential coagulative necrosis of the intestinal mucosa (villi) which extends through the muscularis mucosa and into the submucosa; the necrosis is characterized by loss of differential staining and retention of cellular architecture.  There is multifocal loss of villi and crypts, and blunting and fusion of remaining villi. Bacilli that range in size from 1-2 x 3-7 um line denuded, necrotic villi and form colonies within remaining crypts admixed with necrotic debris.  The lamina propria, submucosa, and serosa are markedly expanded by abundant hemorrhage, increased clear space and ectatic lymphatics (edema), and fibrin. Multifocally, the tunica intima and tunica media of small and medium sized blood vessels are expanded and disrupted by fibrin, edema, karyorrhectic debris, and hemorrhage (necrotizing vasculitis), and occasionally contain organizing eosinophilic, beaded, fibrillar material (fibrin thrombi) which adhere to often disrupted endothelium and either partially or completely occlude the vessel.  Less affected vessels are lined by hypertrophied, reactive endothelium.  The mesentery is edematous and lined by hypertrophied mesothelium.

MORPHOLOGIC DIAGNOSIS:  Small intestine: Enteritis, necrohemorrhagic, acute, diffuse, marked, with vasculitis, edema, and numerous bacilli, Thoroughbred, equine.

ETIOLOGIC DIAGNOSIS:  Clostridial enteritis

CAUSE:  Clostridium perfringens type C

GENERAL DISCUSSION:

• Clostridium perfringens is a Gram positive, sporulating, anaerobic bacillus that has a worldwide distribution and causes disease in neonatal lambs, calves, foals, and piglets (often suckling piglets during the first week of life) and in adult sheep, cattle, goats, horses, and humans
• 7 strains of Clostridium perfringens, designated type A-G based on production of 6 major lethal exotoxins, (alpha, beta, epsilon, iota, CPE, and NetB):
  • Alpha toxin (CPA): Produced by all Clostridium perfringens types; a lecithinase (phospholipase C) that causes cell membrane necrosis and hemolysis; main virulence factor in perfringens type A-associated myonecrosis in humans and animals
  • Beta toxin (CPB): Produced by types B and C; causes necrosis, increases vascular permeability, and decreases mobility of intestinal villi; trypsin labile (i.e. inactivated by trypsin); main factor in type C necrotizing enteritis; tropism for endothelial cells not epithelial
  • Epsilon toxin (ETX): Produced by types B and D during active growth; a necrotizing toxin that forms membrane pores in susceptible cells; causes increased permeability in enterocytes and endothelial cells; produced as a prototoxin and activated by enzymatic (trypsin) digestion in the small intestine; causes hydropericardium, myocardial degeneration and necrosis, myocardial edema, epicardial hemorrhage, pulmonary perivascular/interstitial edema, in addition to the findings of focal symmetric encephalomalacia in sheep
  • Iota toxin (ITX): Produced by type E; causes necrosis and increased vascular permeability; disrupts the cytoskeleton causing cell death; produced as a prototoxin and activated in the small intestine
  • perfringens enterotoxin (CPE): Produced by type F; causes fluid accumulation and mucosal damage; is associated with human food-borne illness most commonly linked to type A; it is activated by proteolysis and alters plasma membrane permeability; opens tight junctions/zona occludens (claudin, occluden, tricellulin, junction-associated molecules [JAM], coxsaxie & adenovirus receptor [CAR])
  • NetB: Produced by type G; causes subclinical necrotic enteritis in chickens, the most important economic outcome from this type
  • NetF: Produced by a recently described toxigenic type A strain (not yet named into its own strain); likely agent responsible for canine acute hemorrhagic diarrhea syndrome and foal necrotizing enteritis; Beta-pore-forming toxin (same family as CPB and NetB), encoded on a plasmid
  • Beta 2-toxin, produced by perfringens type A and type C; is implicated in necrotic enteritis in piglets and typhlocolitis in horses

PATHOGENESIS:

• Change in microenvironment (nutrient rich digesta, antibiotic therapy, altered pancreatic exocrine function, trypsin inhibitors/decreased trypsin levels, other primary infections) allows proliferation of clostridia->colonization of mucus layer-> attach to enterocyte microvilli by Type IV pili (gliding motility)->production of toxins->necrosis of enterocytes and endothelium > hemorrhage, necrosis and edema of lamina propria > death due to diarrhea or secondary bacteremia/toxemia
• Neonates: beta toxin is trypsin labile, and neonates have low trypsin levels which is critical to the pathogenesis; colostrum contains trypsin inhibitors to prevent degradation of colostral antibodies; predisposes to CPB enterotoxemia
• Adult horses may have low trypsin levels due to trypsin inhibitors in feed; decreased pancreatic production of trypsin (e.g. acute pancreatic necrosis, pancreatitis, exocrine pancreatic insufficiency); changes in intestinal microflora; or administration of antibiotics
• In addition to the exotoxins described above, there are 7 minor toxins produced by perfringens include kappa (collagenase); neuraminidase; lambda (caseinase); mu (hyaluronidase); delta (hemolysin); deoxyribonuclease; and urease
• Two zinc metalloproteinases (encoded by zmpA, zmpB) confer additional virulence to perfringens type G in poultry necrotic enteritis

TYPICAL CLINICAL FINDINGS:

• Neonatal (<4-days-old) foals: dehydrated and weak with yellow-brown watery diarrhea, followed by death within 24 hours
• Glycosuria only in lambs with enterotoxemia
• Death often occurs with no clinical signs

TYPICAL GROSS FINDINGS:

• Acute hemorrhagic necrotizing enteritis, particularly of the jejunum and ileum with mucosal necrosis and ulceration surrounded by zone of hyperemia, and free intraluminal blood; intestinal contents may be blood stained; +/- pseudomembrane formation (piglets)
• Peritoneal cavity may contain up to 3 liters of clear, pale yellow fluid, which clots when exposed to air
• Mesenteric lymph nodes are enlarged, congested, and edematous
• Petechial hemorrhages on serous membranes
• Hemorrhages common beneath the epicardium and in the thymus
• Pulmonary interstitial edema

TYPICAL CYTOLOGIC FINDINGS

• Tissue aspirates may show large rods measuring 1 x 4 um, some with a clear, oval, subterminal endospore, and occurring singly or in short chains
• May stain variably due to chronic infection or antibiotic therapy
• Samples often contain abundant cellular debris, lipid, and few inflammatory cells; when neutrophils are present, they are often degenerate

TYPICAL LIGHT MICROSCOPIC FINDINGS:

• Acute necrohemorrhagic enteritis with large numbers of bacilli with square ends (may see sporulation) limited to necrotic tissue, adherent to surface or may be free in lumen
  • May be segmental
  • Hemorrhagic necrosis of intestinal wall may progress to all layers (piglets)
  • Mucosal or submucosal thrombosis
• Mucosal/submucosal hemorrhage
• +/- Fibrinonecrotic pseudomembranes
• +/- Fibrinoid necrotizing vasculitis and thrombosis in submucosa and mucosa
• Usually no inflammation

ADDITIONAL DIAGNOSTIC TESTS:

• CP beta toxin identification in intestinal contents (ELISA) (Gold standard)
• Gram stains
• IHC
• Fecal cytology to determine spore counts and detect bacilli
• Polymerase chain reaction (PCR)
• Anaerobic bacterial culture

DIFFERENTIAL DIAGNOSIS:

Acute diarrhea or sudden death with intestinal necrosis and mucosal thrombosis - horses:

• Salmonellosis - Gram negative bacilli, peracute septicemia in foals; acute fibrinonecrotic ileotyphlocolitis and catarrhal enteritis in horses (similar to perfringens C lesion histologically); chronic necroulcerative typhlocolitis
• Clostridioides difficile - Gram positive bacillus; pseudomembranous typhlocolitis in horses (colitis X), and cats; enterocolitis in foals (similar to perfringens C lesion histologically)

Other equine bacterial enteritides:

• Rhodococcus equi - Gram positive bacillus; pyogranulomatous and necroulcerative typhlocolitis; pyogranulomatous mesenteric lymphadenitis in foals
• Clostridium piliforme (Tyzzer's disease) - Gram negative bacillus; hepatic necrosis; segmental ileotyphlitis/ileocolitis; myocardial necrosis
• Neorickettsia risticci (Potomac horse fever): Diarrhea with congestion and ulceration of  large intestine mucosa with enlarged mesenteric lymph nodes

COMPARATIVE PATHOLOGY:

Clostridium perfringens - Types, toxins, and diseases
Type	Toxin						Diseases
	Alpha	Beta	Epsilon	Iota	cpe	NetB
A	++	-	-	-			Gas gangrene Gangrenous dermatitis in chickens, turkeys Food Borne Illness - humans Gastroenteritis - ferrets Yellow lamb disease - enterotoxemia, western US Colitis horses Canine hemorrhagic diarrhea syndrome (NetF positive strains) Hemorrhagic bowel syndrome - dairy cattle Necrotizing enterocolitis in piglets Antibiotic enteritis- horses, rabbits
B	+	++	+	-			Lamb dysentery Hemorrhagic enteritis - calves, foals, guinea pigs - UK, S. Africa, Middle East Sheep-hemorrhagic enterotoxemia
C	+	++	-	-			Enterotoxic (necro)hemorrhagic enteritis - neonatal lambs, goats, cattle, pigs, foals, occasionally adult horses Struck - Adult sheep, hemorrhagic enteritis and peritonitis due to endothelial damage by angiotoxin Necrotic enteritis of birds Note:  Trypsin (low in neonates) inactivates beta toxin
D	+	-	++	-			Overeating disease/pulpy kidney (due to angiotoxin) - Sheep, cattle, goats (U-B06) Focal symmetric encephalomalacia – Sheep, goats (N-B01) Enterocolitis in goats (not sheep or cattle) Note: Trypsin activates Epsilon toxin
E	+	-	-	++			Enterotoxemia - calves, lambs. guinea pigs, rabbits Canine hemorrhagic gastroenteritis
F	+				+		Human food poisoning and non-foodborne C. perfringens-mediated diarrhea
G	+					+	Poultry necrotic enteritis
Table adapted from Barker et al, 1993 p.237 & Jones et al, 1997 p. 421

• Wild boars (Sus scrofa) can become infected with perfringens types A or C and serve as reservoirs

• Mucoid enteropathy in rabbits may be associated with clostridial overgrowth, though dysautonomia has been documented as a cause

 References: 

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4. Giannitti F, Garcia JP, Rood JI, et al. Cardiopulmonary lesions in sheep produced by experimental acute Clostridium perfringens type D enterotoxemia. Vet Pathol. 2021;58(1):103-113.
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