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 humans7 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 C. 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
C. 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 (Gohari, 2020, JVDI)
Beta 2-toxin, produced by C. perfringens type A and type C; is implicated in necrotic enteritis in piglets and typhlocolitis in horses

Gross and microscopic lesions are not sufficient to differentiate between the five most common causes of enterocolitis in horses; C. perfringens type C, Costridioides difficile, Paeniclostridium sordellii, Salmonella enterica subspecies enterica serovar Typhimurium, and NSAID (Mendonca, 2022, JVDI)

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 C. 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 C. 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
Poultry

Necrotic enteritis- mid-small intestines; distended and friable; mucosa is covered with a brown diphtheritic membrane and contents are foul-smelling, brown fluid

TYPICAL CYTOLOGIC FINDINGS:

Tissue aspirates may show large rods measuring 1 x 4 µm, 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
Phlegmonous gastritis (severe pyogenic bacterial infection of the gastric wall): marked, regionally circumscribed to diffuse expansion of the gastric wall by edema and suppurative inflammation that primarily affects the submucosa, less severely affects the muscularis, and generally spares the mucosa; associated with C. perfringens type C; reported in two yearling horses (Engiles, 2022, JVDI)

ADDITIONAL DIAGNOSTIC TESTS:

CP beta toxin identification in intestinal contents and/or feces (ELISA) (Gold standard)
Gram stains
IHC
Fecal cytology to determine spore counts and detect bacilli; pink safranin counterstain helps distinguish spores
Polymerase chain reaction (PCR); genotyping

Fecal PCR panels (Magdesian, 2022, JVDI)

Anaerobic bacterial culture

Isolation of C. perfringens type C from animals with necrotizing enteritis is highly suggestive of infection because this bacteria is rarely present in the intestine of healthy animals (Uzal, 2022, JVDI)

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 C. 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 C. 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
Type	Alpha	Beta	Epsilon	Iota	cpe	NetB	Diseases
A	++	-	-	-			Gas gangrene Gangrenous dermatitis - 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 - piglets Necrotic enteritis – toucans Antibiotic enteritis - horses, rabbits Postpartum clostridial gangrenous metritis – dairy goats
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); there are no specific gross or histologic renal lesions found in sheep with experimental acute type D enterotoxemia (Giannitti, 2023, Vet Pathol) Focal symmetric encephalomalacia – Sheep, goats (N-B01) Cardiopulmonary disease – sheep Enterocolitis in goats (not sheep or cattle) Note: Trypsin activates Epsilon toxin; some isolates may also code for lambda toxin which can cleave ETX and cause disease in neonatal lambs and kids (Acevedo, 2023, JVDI)
E	+	-	-	++			Enterotoxemia - calves, lambs. guinea pigs, rabbits Canine hemorrhagic gastroenteritis
F	+				+		Human food poisoning and non-foodborne C. perfringens-mediated diarrhea Necrotic enteritis – turkeys
G	+					+	Necrotic enteritis - poultry
Table adapted from Barker et al, 1993 p.237 & Jones et al, 1997 p. 421

    Wild boars (Sus scrofa) can become infected with C. 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; C. perfringens causes cecal hemorrhage and edema in young rabbits
Clostridium perfringens was one of the three most common pathogens found to cause enteritis or enterocolitis in Wisconsin white-tailed deer; other two are rotavirus and E. coli (Clarke, 2023, JVDI)
Bats: Hemorrhagic diarrhea (European vespertilionid bats) and gastritis (captive pteropodid bats)
Nonhuman primates: Acute gastric dilation or “bloat-syndrome”; overeating and drinking, following anesthesia; death usually due to shock and pressure on the thoracic organs and vascular system
Update (differs from other required reading): Gross and microscopic lesions, culture, and sequencing results were not supportive of C. perfringens or Aspergillus fumigatus as the cause for hemorrhagic bowel syndrome (De Jonge, 2023, Vet Pathol)

    Chickens: Clostridium perfringens (necrotic enteritis) must be carefully differentiated from Clostridium colinum (ulcerative enteritis); coccidiosis can predispose poultry to C. perfringens

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