JPC SYSTEMIC PATHOLOGY
DIGESTIVE SYSTEM
September 2021
D-P06

 

 

SLIDE A: Signalment (JPC #4161728):  10 year old, male, corn snake

 

HISTORY:  Presented with a six month history of intermittent regurgitation and weight loss and was subsequently euthanized.

 

HISTOPATHOLOGIC DESCRIPTION:  Stomach, glandular portion:  Diffusely the gastric mucosa is thickened up to 4mm, forming long papillary and frond-like projections up to 6 mm in length (hyperplasia).  There are increased numbers of mucous cells that often replace, separate, or compress gastric glandular epithelium (mucus hyperplasia and metaplasia).  Lining the brush border of gastric epithelium and free within the lumen are numerous 2-6 um diameter, round, pale amphophilic to basophilic apicomplexan protozoa/protists with variably distinct 1-2 um basophilic nuclei.  Multifocally within the stomach lumen and expanding the gastric gland lumina are sloughed epithelial cells, rare macrophages, heterophils, and large aggregates 1-3 um coccobacilli in amphophilic, fibrillar fluid.  Multifocally the lamina propria contains small numbers of lymphocytes, plasma cells, and fewer heterophils.  Multifocally, within the muscularis externa predominately at the junction of the circular and longitudinal layers often surrounding vessels and myenteric plexus are aggregates of numerous lymphocytes, fewer macrophages, and heterophils. Multifocally, the gastric serosa is thickened up to 5-7 times normal and is expanded by edema, fibrin, lymphocytes, and rare plasma cells.  Multifocally, within the submucosa, lymphatics are etatic and filled with a pale eosinophilic fluid (edema).

 

MORPHOLOGIC DIAGNOSIS:  Stomach:  Gastritis, proliferative and lymphoplasmacytic, chronic, diffuse, marked, with mucus neck cell hyperplasia and metaplasia and numerous free and apically attached apicomplexans, etiology consistent with Cryptosporidium sp., corn snake (Pantherophis guttatus), ophidian.

 

ETIOLOGIC DIAGNOSIS:  Gastric cryptosporidiosis

 

CAUSE:  Cryptosporidium serpentis

 

SLIDE B: Signalment (JPC #3103340): 4-year-old intact male rhesus macaque

 

HISTORY: Animal was CD8 depleted, treated with anti-IL-15 monoclonal antibody, and infected with simian immunodeficiency virus (SIV) to study CD4-T cell kinetics. Months after infection, animal became lethargic and anorexic with weight loss.

 

HISTOPATHOLOGIC DESCRIPTION: Bile duct, extrahepatic: There is marked circumferential biliary epithelial hyperplasia, characterized by piling up of epithelial cells with formation of prominent villar-like papillary projections that extend into the lumen. The mucosal epithelium ranges from cuboidal to columnar with large, prominent, vesiculate nuclei, increased mitotic figures (up to three per HPF [0.237mm²]) and scattered apoptotic cells. Goblet cells are increased in number and multifocally replace epithelial cells (goblet cell hyperplasia and metaplasia).  Lining the epithelial brush border and free within the lumen are many 2-6 um diameter, round, pale amphophilic to basophilic apicomplexan protozoa/protists with variably distinct 1-2 um basophilic nuclei.  Multifocally the lamina propria contains moderate numbers of lymphocytes, plasma cells, and fewer histiocytes and eosinophils admixed with mild hemorrhage that extends multifocally into the adjacent mesentery. There is mild periductal fibrosis.  Multifocally expanding the mesenteric fibrous connective tissue is abundant edema, fibrin, and scant hemorrhage.

 

MORPHOLOGIC DIAGNOSIS: Bile duct: Cholangitis, proliferative, chronic, circumferential, moderate, with goblet cell hyperplasia and metaplasia, periductal fibrosis and apical apicomplexans, etiology consistent with Cryptospordium sp., rhesus macaque (Macaca mulatta), nonhuman primate.

 

ETIOLOGIC DIAGNOSIS: Biliary cryptosporidiosis

 

CAUSE: Cryptosporidium sp.

 

GENERAL:

• Cryptosporidium sp. are highly infectious, 2-8 um diameter, apicomplexan protozoa that colonize the surface epithelium of the gastrointestinal, biliary, and respiratory tracts of mammals, birds, reptiles, and fish
• The protozoa displace the microvilli and are enclosed by surface cell membranes, thus described as intracellular but extracytoplasmic
• Respiratory disease is most significant in birds while enteric disease predominates in other vertebrates
• To date there are 20 recognized Cryptosporidium with nearly 61 genotypes

 

LIFE CYCLE:

• Sporulated oocysts are inhaled or ingested; sporozoites are released (excystation) and attach to the apical brush border of enterocytes covering tips and sides of villi, where merogony (asexual), gametogony (sexual), and sporogony occur
• Type I meronts produce up to 8 merozoites, which recycle to form more type I schizonts, or produce type II meronts; type II meronts have 4 merozoites that invade the host cell and undergo gametogony
• Two kinds of oocysts formed: Thin-walled and thick-walled; they sporulate within the host cell
• Autoinfection occurs via thin-walled oocysts; thick-walled oocysts are shed in the nasal secretions and feces

 

PATHOGENESIS:

• In snakes, Cryptosporidium causes reduction in acid-secreting granular cells with replacement by mucus neck cells (mucous metaplasia and hyperplasia) resulting in gastric mucosal hypertrophy
• Cryptosporidium-associated diarrhea develops in mammals primarily due to malabsorption associated with severe villous atrophy, immature enterocytes occupied by high numbers of organisms reducing absorptive surface area, and release of inflammatory mediators, principally prostaglandins, that alter mucosal secretion and electrolyte and macromolecule transport
• Primary infection with another agent often causes immunodeficiency which predisposes animal to secondary infection with Cryptosporidium
• IFN- γ is important for resistance to and clearance of the infection and is a major mediator in host resistance

 

CLINICAL FINDINGS:

• In snakes:
  • Postprandial regurgitation 1-3 days after eating
  • Palpably firm stomach that may cause the surrounding body wall to bulge
  • Progressive emaciation without loss of appetite
• In mammals: Diarrhea (subacute or chronic, sometimes bloody, watery)

 

GROSS FINDINGS:

• Gastric wall often more than a centimeter thick even in small snakes
• Markedly reduced lumen
• Frequently abnormal amount of mucus adherent to the mucosa

 

MICROSCOPIC FINDINGS:

• Numerous organisms attached to brush border of epithelial cells

Gastric lesions in snakes:

• Metaplasia and hyperplasia of mucus neck cells of the gastric glands
• Replacement of the granular cells by mucus cells
• Edema of lamina propria and muscular tunics
• +/- inflammation and necrosis

Intestinal lesions in mammals:  

• Villus atrophy with blunting and fusion and
• hypertrophy and hyperplasia of crypt epithelium
• One study identified organisms in the epithelium of the common bile duct, the intrahepatic and pancreatic ducts, and the gallbladder of a monkey

 

ULTRASTRUCTURAL FINDINGS:

• Organism attaches to enterocytes, displacing microvilli
• Enclosed within double layered invagination of host cell membrane on apical
• surface thus residing intracellularly but extracytoplasmic within a parasitophorous vacuole
• Feeder organelle is present at the base of attachment between the parasitophorous
• vacuole and cytoplasm of host cell

 

ADDITIONAL DIAGNOSTIC TESTS:

• Snakes: Demonstrate organisms after gastric lavage
• Other animals: Fecal flotation or respiratory fluids
• Histochemical stains: modified acid fast stain, Giemsa, merthiolate, Mayer’s
• hematoxylin
• Fluorescent staining with auramine O or fluorescein-labeled antibody

 

DIFFERENTIAL DIAGNOSIS:

• Enterocytozoon bieneusi: microsporidian parasite that may infects the intestine, liver or gall bladder of new world monkeys and may easily be mistaken for Cryptosporidium; 
  • E. bieneusi has birefringent intracellular spores and is Gram positive
  • Cryptosporidium does not stain with gram stain and is located in the brush border and lumen

 

COMPARATIVE PATHOLOGY:

• Cryptosporidium with gastric localization:
  • C. andersoni (bovine abomasums, larger usually 6-8 microns)
  • C. muris (gastric gland- mice, gerbils, hamsters, other mammals)
  • C. muris-like organism (gastric glands- cynomolgus monkeys)
  • C. fragile (amphibians)
  • C. serpentis (snakes)
  • C. varanii (reptiles)
• Cryptosporidium with intestinal localization:
  • C. parvum (ruminants, horses, dogs, gerbils, hamsters, mice, and numerous wild animals)
    • Suckling calves are the main host of parvum
    • In a study, infection with C. parvum induced mastocytosis in the mucosa of the entire small intestine in immune competent calves (Li, Vet Pathol. 2013)
  • C. hominis (humans, nonhuman primates)
  • C. Cbovis (calves)
  • C. canis (dogs)
  • C. felis (cats)
  • C. suis (pigs)
  • C. ryanae (bovine is only know host of this species)
  • C. wrairi (guinea pigs – associated with clinical cases of E. coli)
  • C. fayeri, macropodum (marsupials)
  • C. cuniculus (rabbits) – zoonotic significance
• Birds: baileyi and C. meleagridis are most common species in chickens, quails, and turkeys, causing intestinal (and cloaca) and respiratory infections; also associated with infectious bursal disease (avian birnavirus); one report of renal and cloacal cryptosporidiosis (C. avian genotype V) in a Major Mitchell's cockatoo (Lophochroa leadbeateri) (Curtiss, J Zoo Wildl Med. 2015)
  • In Ostrich chicks (especially males), cryptosporidosis can lead to cloacal prolapse due to mucosal swelling and epithelial hyperplasia in the distal rectum/cloaca
• Reptiles: sC. erpentis, C.varanii;
  • C. varanii reported in lizards, snakes, and other reptilian species 
  • C. dusismarci recently reported for the first time in a captive African spurred tortoise (Rostad, J Comp Pathol. 2019)
  • Cryptosporidium infections have been associated with ear lesions (aural-pharyngeal polyps) in green iguanas
  • Report of 16 wild green snakes with cryptosporidium infections; did not have the classic gastric mucosal hypertrophy but enteritis only as compared to their captive counterparts (Brower, J Zoo Wildl Med. 2001)

• Amphibians: fragile; reported in black-spined toads
• Fish: molnari, molnari-like species
• Marine mammals: known to harbor Cryptosporidum (especially with spp. that are commonly associated with humans.
• Numerous Cryptosporidium sp. are zoonotic (mainly C. parvum and to a lesser extent C. suis, C. canis, and C. felis); several cases reported in animal handlers caring for infected infant nonhuman primates in a nursery
• Severe fatal infections occur in Arabian foals with SCID; humans and monkeys infected with HIV and SIV, respectively; and humans with drug-induced immunosuppression; parvum was responsible for the largest waterborne outbreak of diarrhea in U.S. history

 

REFERENCES:

1. Barthold SW, Griffey SM, Percy DH. Pathology of Laboratory Rodents and Rabbits. 4^th Ames, IA: John Wiley and Sons, Inc; 2016: 82, 108, 152, 189, 204-205, 235-236, 297.
2. Brower AI, Cranfield MR. Cryptosporidium-associated enteritis without gastritis in rough green snakes (Opheodrys aestivus) and a common garter snake (Thamnophis sirtalis). J Zoo Wildl Med. 2001;32(1):101-105.
3. Curtiss JB, Leone AM, Wellehan JF Jr, Emerson JA, Howerth EW, Farina LL. Renal and cloacal cryptosporidiosis (Cryptosporidium avian genotype V) in a Major Mitchell’s cockatoo (Lophochroa leadbeateri). J Zoo Wildl Med. 2015;46(4):934-937.
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