JPC SYSTEMIC PATHOLOGY

DIGESTIVE SYSTEM

September 2024

D-F03

 

SIGNALMENT (JPC #1632089): An adult Vizsla dog.

 

HISTORY: This dog presented with intractable diarrhea unresponsive to therapy. 

 

HISTOPATHOLOGIC DESCRIPTION: SLIDE A: Colon: Within the lamina propria, muscularis mucosa, submucosa, serosa, and mesentery, there are abundant extracellular and intrahistiocytic, round to oval, 8-20 µm diameter algal sporangia that have a clear, 2-4 µm thick, anisotropic wall and contain either central granular amphophilic material or multiple (2-8 or more) wedge-shaped endospores. These algae are surrounded by multifocal to coalescing inflammatory infiltrates composed of moderate numbers of macrophages and plasma cells, and rare lymphocytes that moderately expand the lamina propria, extend through the muscularis mucosa, and expand the submucosa up to twice normal thickness. Leiomyocytes within the muscularis mucosa are multifocally swollen with pale eosinophilic vacuolated sarcoplasm (degeneration). There is congestion of submucosal blood vessels and there is multifocal mild hemorrhage, fibrin, and edema. The serosa and mesentery are multifocally expanded by previously described algae and inflammatory cells which separate and surround adipocytes (steatosis). Rarely, the superficial mucosal epithelium is disrupted by eosinophilic cellular and karyorrhectic debris (lytic necrosis). 

 

SLIDE B: Colon (PAS): The algal cell wall is PAS positive. 

 

MORPHOLOGIC DIAGNOSIS: Colon: Colitis, histiocytic and plasmacytic, subacute, multifocal to coalescing, moderate, with mesenteric steatitis and abundant extracellular and intrahistiocytic algae, Vizsla, canine.

 

ETIOLOGIC DIAGNOSIS: Protothecal colitis

 

CAUSE: Prototheca spp.  

 

GENERAL DISCUSSION: 

• Colorless, unicellular, opportunistic algae; reproduces asexually via endosporulation resulting in septation of the parent cell (sporangium); often forms the classic "Mercedes Benz" symbol
• Causes enteric, cutaneous, or generalized granulomatous disease in various mammals 
• Causes gastrointestinal or disseminated infections in dogs, cutaneous infections of cats and humans, and mastitis in cows
• At least seven species; five cause disease: Prototheca zopfii, P. wickerhamii, P. blaschkeae, and P. miyajii, and P. salmonis

• Until 2008, only P. zopfii and P. wickerhamii were known to be pathogenic in humans and mammals (most common)

 

PATHOGENESIS: 

• Pathogenesis and predisposing factors are not completely understood
• Prototheca sp. most commonly isolated from sewage and animal waste > contamination of water systems, soil, and food > initiates infection via traumatic inoculation in cutaneous form, invasion of teat canal in mammary form, and penetration of colonic mucosa (disseminated form) > depressed cell-mediated immunity, in the dog, allows opportunistic invasion and circulatory dissemination
• Collies and boxers may be over-represented, suggesting breed-related susceptibility
• Altered or suppressed host immune response is an important factor; cell-mediated immunity vital to eliminate infection

 

TYPICAL CLINICAL FINDINGS:

• Dogs: chronic, episodic, intractable, hemorrhagic large-bowel diarrhea
• In disseminated disease, signs depend on tissues affected (central nervous system, kidneys, heart, eyes (S-M03) most frequently involved)
• Ophthalmic signs include acute mydriasis, blindness, chorioretinitis, retinal detachment, anterior uveitis, endophthalmitis
• Central nervous system signs (including blindness and seizures) occur in 40-60% of cases and often follow gastrointestinal signs

TYPICAL GROSS FINDINGS: 

• Hemorrhagic and ulcerative colitis is first and most consistent enteric lesion
• Mesenteric lymph node enlargement

• Intestine: Small white to gray serosal nodules; friable, thickened corrugated colon/rectum; patchy hemorrhagic necroulcerative enterocolitis

• Kidney: Disseminated white to gray nodules or surface depressions, with pale foci within parenchyma and at corticomedullary junction; necrosis of the renal papilla
• Liver: Enlarged, lobulated; patchy yellow discoloration
• Eye: Exudate in anterior chamber; gray-white granular material in vitreous
• Serosa: Disseminated gray nodules (peritoneum, pericardium)

 

TYPICAL LIGHT MICROSCOPIC FINDINGS: 

• Cellular host inflammatory response to infection can vary; granulomatous to pyogranulomatous ulcerative colitis is typical
• Intracellular and extracellular algae:
  • Difficult to visualize; PAS or silver stains helpful
  • Unicellular, nonbudding, round to oval spherules with a refractile capsule
  • Range in size from 5-12µm (P. wickerhamii) to 10-25µm (P.zopfii)
  • Multiple (2 to 20) wedge-shaped endospores (sporangiospores) within a sporangium is characteristic
• Cytologic findings:
  • Usually identified in samples from large intestine or feces
  • Oval to kidney bean shaped, 1-14µm wide and 1-16µm long, with granular basophilic cytoplasm, and a thin clear cell wall
  • If mature, contain small pink to deep purple nucleus
  • Multiple basophilic round structures (endospores) occurring singly and in clusters
  • Empty casings (theca)

 

ULTRASTRUCTURAL FINDINGS:

• “Mercedes-Benz” endospores – tripartite cell wall division

• Prototheca sp: Central nucleus, large nucleolus; thick granular cell wall; electron dense bodies in cytoplasm; starch granules; no chloroplasts
• Chlorella sp: Distinct cell wall coating (Sporopollenin layer); thick cell wall; chloroplasts (arranged in 3-5 thylakoid stacks) surrounding electron dense starch granules and small plastoglobules

ADDITIONAL DIAGNOSTIC TESTS: 

• Antemortem diagnosis with CSF cytology and PCR (Asiag, J Vet Diagn Invest. 2022)
• Culture (easily cultured on fungal media without cycloheximide)
• Unstained wet mounts, cytology
• Romanowsky type stains: Wrights, Giemsa
• Fungal stains: periodic acid Schiff (PAS), Gridley, or Gomori methenamine silver (GMS) 
• Others: fluorescent antibody test, electron microscopy, PCR

 

DIFFERENTIAL DIAGNOSIS:

Organisms that reproduce by endosporulation (mnemonic CRCB):

• Chlorella spp.: Nearly impossible to differentiate from Prototheca sp. in H&E stained sections with standard light microscopy, but tissue is green in color grossly; Chlorella sporangia contain birefringent cytoplasmic starch bodies and chloroplasts stained by Gridley, GMS, and PAS that are PAS-negative after diastase digestion; reported in cattle, sheep, gazelle, and beaver; chloroplasts are evident on EM; 
• Rhinosporidium seeberi: Nasal mucosa; large, mature sporangia 100-350 um diameter with numerous round, mature endospores 7-9 um diameter
• Coccidioides immitis: Larger (10‑60 um), with many endospores per sporangium
• Batrachochytrium dendrobatidis (Chytridiomycosis): Amphibian disease that incites epithelial hyperplasia/hyperkeratosis; thalli with discharge papillae in the epidermis
• Helicosporidium sp.: Will be PAS, GMS, and acid-fast positive (Asin, Vet Pathol. 2024)

 

Organisms with similar histologic appearance:

• Cryptococcus – Narrow base budding; thick polysaccharide capsule
• Blastomyces – Broad based budding

 

Granulomatous colitis in dogs:

• Histoplasma capsulatum: Ulcerative, granulomatous transmural colitis
• Pythium insidiosum: Eosinophilic and granulomatous colitis
• Trichuris vulpis (whip worms): Presence of intraluminal nematodes; usually 

affects cecum and proximal ascending colon; evolves into a granulomatous transmural condition

• Mycobacterium spp. (M. bovis, M. tuberculosis)  
• Leishmania sp: Heavy mucosal infiltrate of macrophages and often plasma cells

 

COMPARATIVE PATHOLOGY: 

• Cats: Single or multiple, firm, small-to-large, gray-white or tan cutaneous to subcutaneous nodules most commonly on limbs and feet (other sites include head, pinna, and base of tail); lesions may extend deeply into fascia, tendons, vessels, and nerves +/- regional lymph nodes; dense infiltrations of macrophages and giant cells without discrete granulomas
• Bovine: Nodular granulomatous mastitis with massive necrotic areas, thrombosis, and hemorrhage; enlarged and pale supramammary lymph node; giant cells and eosinophils common; P. blaschkeae isolated from mammary gland of cows with mastitis (Riet-Correa, J Vet Diagn Invest. 2021)
• Horses: Rhinitis
• Goats: Cutaneous and nasal protothecosis (Riet-Correa, J Vet Diagn Invest. 2021)
• Humans (3 forms): Cutaneous protothecosis (most prevalent); olecranon bursitis; disseminated; P. miyajii isolated from human patient with systemic disease (Riet-Correa, J Vet Diagn Invest. 2021) Flying fox: Disseminated granulomatous disease in captive flying fox
• California kingsnake (Lampropeltis californiae): Closely related genus of nonphotosynthetic, green algae Helicosporidium causes systemic disease (Asin, Vet Pathol. 2024)

 

REFERENCES:

1. Asiag N, Lapid R, Aizenberg Z, Baneth G, Nachum-Biala Y, Leszkowicz-Mazuz M, Yasur-Landau D, Chai O, Aroch I, Shamir MH. Spinal cord protothecosis causing paraparesis in a dog. J Vet Diagn Invest. 2022;34(4):684-688.
2. Asin J, Childress AL, Dervas E, Garner MM, Uzal FA, Wellehan JFX Jr, Henderson EE, Armien AG. Helicosporidium sp. infection in a California kingsnake (Lampropeltis californiae): Spillover of a pathogen of invertebrates to a vertebrate host. Vet Pathol. 2024: online.
3. Boes KM. Chapter 6: Body Cavity Fluids. In: Raskin RE, Meyer DJ, & Boes KM eds. Canine and Feline Cytopathology: A Color Atlas and Interpretation Guide. 4th ed. St. Louis, MO: Elsevier; 2022:244, 258.
4. Falcaro C, Furlanello T, Binanti D, et al. Molecular characterization of Prototheca in 11 symptomatic dogs. J Vet Diagn Invest. 2021;33(1):156-161.
5. Farina LL, Lankton JS. Chiroptera. In: Terio KA, McAloose D, St. Leger J ed. Pathology of Wildlife and Zoo Animals. Cambridge, MA: Elsevier Inc. 2018:626.e3.
6. Haddad JL, Marks Stowe DA, Neel JA. The Gastrointestinal Tract. In: Valenciano AC, Cowell RL, eds. Diagnostic Cytology and Hematology of the Dog and Cat. 5th ed. St. Louis, MO: Elsevier Mosby; 2014:314.
7. Lane LV, Yang PJ, Cowell RL. Selected Infectious Agents. In: Valenciano AC, Cowell RL, eds. Diagnostic Cytology and Hematology of the Dog and Cat. 5th ed. St. Louis, MO: Elsevier Mosby; 2014:61. 
8. Mauldin EA, Peters-Kennedy J. Integumentary system. In: Maxie MG, ed. Jubb, Kennedy, and Palmer’s Pathology of Domestic Animals. Vol 1. 6th ed. St. Louis, MO: Elsevier; 2016:665.
9. Raskin RE, Conrado FO. Chapter 3: Integumentary System. In: Raskin RE, Meyer DJ, & Boes KM eds. Canine and Feline Cytopathology: A Color Atlas and Interpretation Guide. 4th ed. St. Louis, MO: Elsevier; 2022:34-122.
10. Riet-Correa F, Carmo PMSD, Uzal FA. Protothecosis and chlorellosis in sheep and goats: a review. J Vet Diagn Invest. 2021;33(2):283-287. 
11. 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; 2022:454. 
12. Stanton JB, Zachary JF. Mechanisms of Microbial Infections. In: Zachary JF, ed. Pathologic Basis of Veterinary Disease. 7th ed. St. Louis, MO: Elsevier; 2022:254. 
13. Uzal FA, Plattner BL, Hostetter JM. Alimentary System. In: Maxie MG, ed. Jubb, Kennedy and Palmer’s Pathology of Domestic Animals. 6th ed. Vol 2. St. Louis, MO: Elsevier; 2016:203-204. 
14. Wilcock BP, Njaa BL. Special senses. In: Maxie MG, ed. Jubb, Kennedy, and Palmer’s Pathology of Domestic Animals, Vol 1. 6th ed. St. Louis, MO: Elsevier; 2016: 451.

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