JPC SYSTEMIC PATHOLOGY
DIGESTIVE SYSTEM
August 2021
D-F03

 

 

SIGNALMENT (JPC #1632089):  An adult Vizsla dog

 

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

 

SLIDE A: HISTOPATHOLOGIC DESCRIPTION:  Colon:  Within the lamina propria, muscularis mucosa, submucosa, serosa, and mesentery, there are abundant extracellular and intrahistiocytic, round to oval, 8-20 um diameter algal sporangia that have a clear, 2-4um thick, anisotropic wall and contain either central granular amphophilic material or multiple (2-8 or more) wedge-shaped endospores.  These are surrounded by multifocal to coalescing inflammatory infiltrates composed of moderate numbers of macrophages and plasma cells, and rare lymphocytes that diffusely moderately expand the lamina propria, extend through the muscularis mucosa, and expand the submucosa up to twice normal thickness.  Leiomyocytes within the muscularis mucusa are multifocally swollen with lightly eosinophilic vacuolated sarcoplasm (degeneration).  There is congestion of submucosal blood vessels and there is multifocal mild submucosal hemorrhage, fibrin, and edema. The serosa and mesentery are multifocally expanded by algae and previously described inflammatory cells which separate and surround adipocytes (steatosis).  Rarely, the superficial mucosal epithelium is disrupted by eosinophilic cellular and karyorrhectic debris (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, saprophytic algae; reproduces asexually via endosporulation resulting in septation of the parent cell (sporangium); often forms the classic "Mercedes Benz" symbol
• Causes enteric, cutaneous, ocular (see S-M03), nasal, mammary, neurologic, or generalized granulomatous diseases 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 miyajii, and P. salmonis
  • Until 2008, only P. zopfii and P. wickerhamii were known to be pathogenic in humans and mammals (most common)

• Closely related to the green algae of genus Chlorella; Prototheca lack chlorophyll

 

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 contact with cutaneous wounds (cutaneous form) and penetration of intestinal 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 or immunodeficiency
• Bovine mammary form is thought to occur by environmental contamination and ascending infection
• Altered or suppressed host immune response is an important factor, and defective cell-mediated immunity appears to be more important than decreased humoral response in allowing entrance of organisms

 

TYPICAL CLINICAL FINDINGS:

• Generally a widely disseminated disease, with signs depending on tissues affected (kidney/urinary tract, liver, heart, intestine, brain, and eye(S-M03) most frequently involved)
• Hemorrhagic and ulcerative colitis is first and most consistent enteric lesion
• Ophthalmic signs include acute mydriasis, blindness, chorioretinitis, retinal detachment, anterior uveitis, endophthalmitis
• Most frequently reported sign is bloody diarrhea, concurrent with weight loss and progressive debility
• Central nervous system signs (including blindness and seizures) occur in 40-60% of cases and often follow gastrointestinal signs

TYPICAL GROSS FINDINGS: 

• 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
• Algae:
  • Occur as either single endospores or as large sporangia containing multiple endospores; presence of multiple wedge-shaped endospores within a single sporangium is characteristic of Prototheca (wedge-shaped, radially arranged, angular endospores (daughter cells) separated by septations fill sporangia)
    • P. zopfii is usually larger (10-25 microns), with oval sporangia containing round to polyhedral endospores
    • P. wickerhamii is usually smaller (5-12 microns), with characteristic sporangia with a central rounded endospore surrounded by a corona of molded endospores which is described as moruloid, daisy-like, spoke-like, and frambesiform
  • May be extracellular or intraphagocytic
  • Cells are ovoid with a thick, granular, refractile, PAS positive, and argyrophilic cellulosic cell wall and granular, weakly basophilic cytoplasm surrounding a central nucleus

 

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: 

• Culture (easily cultured on fungal media without cycloheximide)
• Unstained wet mounts
• 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 : 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 many large starch granules stained by Gridley, GMS, and PAS; 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

 

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; 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; miyajii isolated from human patient with systemic disease (Riet-Correa, J Vet Diagn Invest. 2021)
• Salmon: Renal granulomatous disease ( salmonis)

 

REFERENCES:

1. Chengappa MM, Pohlman LM. Agents of systemic mycosis. In: McVey DS, Kennedy M, Chengappa MM eds. Veterinary Microbiology. 3^rd Ames, IA: John Wiley & Sons, Inc; 2013:343.
2. Cheville NF. Algae, fungi and other eukaryotes. In: Ultrastructural Pathology: The Comparative Cellular Basis of Disease. 2nd ed. Ames, IA: Wiley-Blackwell; 2009:573-576.
3. 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.
4. 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.
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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. Pressler BM. Protothecosis and Chlorellosis. In: Greene CE, ed. Infectious Diseases of the Dog and Cat. 4th ed. St. Louis, MO:Elsevier; 2012:696-701.
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. 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.
12. 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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