JPC SYSTEMIC PATHOLOGY
SPECIAL SENSES
April 2021
S-M03 (NP)

Signalment (JPC #4002461):  5-year-old female spayed Rhodesian ridgeback, canine

HISTORY: The dog was presented for a large (approximately 7 cm diameter), subcutaneous moveable mass over the left shoulder. Two weeks after surgical excision, the dog presented with neurologic signs and blindness. Euthanasia was elected because of poor prognosis.

HISTOPATHOLOGIC DESCRIPTION:  Eye:  Focally extensively elevating the diffusely detached retina and multifocally extending into the subjacent choroid is a densely cellular exudate composed of many viable and degenerate neutrophils and macrophages admixed with eosinophilic cellular and karyorrhectic debris (necrosis), fibrin, eosinophilic globular protein, and minimal hemorrhage and mineral.  There are many round algal sporangia both free within the exudate as well as within macrophages. These sporangia are 10-20 um in diameter with a 2-3 um thick amphophilic cell wall and a basophilic nucleus.  Many algae undergo endosporulation in which the sporangia are filled by wedge-shaped, radially arranged, angular endospores (daughter cells) divided by septations.  The detached retina is thickened by fibrin, hemorrhage, and edema, and multifocally few vessel walls within the retina are expanded by few neutrophils, brightly eosinophilic protein, cellular debris (vasculitis), and are surrounded by few lymphocytes and histiocytes.  There is marked asymmetrical necrosis and loss of varying layers of the retina, most prominently adjacent to areas of exudate.  There is moderate hyperplasia and hypertrophy of the retinal pigment epithelium.  The choroid and ciliary body are multifocally infiltrated and/or expanded by few to many neutrophils, macrophages, lymphocytes, plasma cells, congested vessels, and dilated lymphatics.  Multifocally within the sclera, lymphatics are dilated and filled with edema and mild hemorrhage and inflammatory cells.

MORPHOLOGIC DIAGNOSIS:  Eye:  Endophthalmitis, pyogranulomatous, diffuse, moderate, with exudative retinal detachment and necrosis, and numerous extracellular and intrahistiocytic endosporulating algae, Rhodesian ridgeback, canine.

ETIOLOGIC DIAGNOSIS:  Ocular protothecosis

CAUSE:  Prototheca zopfii

GENERAL DISCUSSION:

• Prototheca are colorless, unicellular, saprophytic algae capable of causing enteric (see D-F03), cutaneous, nasal, mammary, neurologic, or generalized granulomatous diseases in various mammals
• Of the five species of Prototheca, only zopfii and P. wickerhamii cause disease in animals; mastitis in cows and disseminated disease in dogs are more common manifestations
• Closely related to the green algae of genus Chlorella
• Algae are regarded as opportunistic pathogens of low pathogenicity usually affecting animals with immune dysfunction
• In dogs, the organisms have a predilection for the eyes and brain
• In other species, infections are commonly localized to the skin or subcutis
• Prototheca reproduces by endosporulation resulting in septation of the parent cell forming the classic "Mercedes Benz" symbol
• The virulence of the two species of pathogenic Prototheca may differ:
  • Only wickerhamii has been isolated in cutaneous cases of disease in dogs and cats
  • Disseminated disease in dogs is almost always caused by zopfii
    • Ocular lesions been described only in dogs with disseminated form
    • Lesions are similar those of blastomycosis and cryptococcosis
  • A disproportionate number of cases have been reported in boxer (females) and collies (both breeds overrepresented in some reports)

PATHOGENESIS:

• The pathogenesis is not completely understood and factors predisposing to developing intestinal protothecosis are poorly 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
• 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:

Dogs: 

• Generally a widely disseminated disease, with signs depending on tissues affected (kidney/urinary tract, liver, heart, intestine, brain, and eye most frequently involved)
• Ophthalmic signs include acute mydriasis, blindness, chorioretinitis, retinal detachment, anterior uveitis, endophthalmitis
• Hemorrhagic and ulcerative colitis is first and most consistent enteric lesion
• 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

Cats: 

• Disease is typically manifested as large, firm, cutaneous nodules most commonly on limbs and feet; other sites include head, pinna, and base of tail
• Cats are otherwise generally healthy

Cattle: 

• Massive necrotic lesions in udder (mastitis) with enlarged local lymph nodes

TYPICAL GROSS FINDINGS:

Dogs: 

• White to tan, nodular lesions in gastrointestinal tract, eye, heart, kidney, liver, etc.
• Cloudy exudate in the chambers of the eye and between detached retina and choroid

Cats:

• Localized gray-white, subcutaneous or dermal lesions that extend deeply into fascia, tendons, vessels, and nerves

Cattle: 

• Mastitis and regional lymphadenitis; massive necrosis of udder, with thrombosis and hemorrhage

TYPICAL LIGHT MICROSCOPIC FINDINGS:

Dogs: 

• Eye: Bilateral lesions; vary from lymphoplasmacytic to granulomatous panuveitis with optic neuritis and exudative retinal separation

Cats: 

• Cutaneous granulomas, with numerous epithelioid macrophages and few multinucleated giant cells

Cattle: 

• Reaction varies from extensive, acute, necrotizing lesions with minimal inflammatory reaction to granulomatous and pyogranulomatous mastitis and lymphadenitis with or without the presence of eosinophils
• Vasculitis, thrombosis, and many algal organisms

Histomorphology of Algae:

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

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
• 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:

• Vitreous centesis
• GMS, PAS, Gridley (GF) special stains

DIFFERENTIAL DIAGNOSIS:

Endosporulating organisms:

• Chlorella: Very similar to Prototheca sp, but tissue is green in color grossly; Chlorella sporangia contain many large starch granules stained by Gridley, Gomori Methenamine Silver, and Periodic acid-Schiff; reported in cattle, sheep, gazelle and beaver; granulomatous and necrotizing lesions with similar organisms containing intracytoplasmic, irregular, birefringent starch granules which are PAS positive-diastase sensitive, anisotropic in unstained and H&E sections and chloroplasts are evident on EM; nearly impossible to differentiate in H&E stained sections with standard light microscopy
• Coccidioides immitis: Larger (10‑60 um), with many endospores per sporangium
• Rhinosporidium seeberi: Large, distinctive organisms in nasal mucosa
• Chytridiomycosis (Batrachochytrium dendrobatidis): Amphibian disease that incites hyperplastic and hyperkeratotic epithelial lesions
• Cryptococcus – Narrow base budding; thick polysaccharide capsule
• Blastomyces – Broad based budding

COMPARATIVE PATHOLOGY:

• Salmon: salmonis causes renal granulomatous disease of salmon
• Cattle: zopfii causes mastitis 
• Deer: Pustular dermatitis
• Goat: Ulcerative and granulomatous dermatitis
• Miscellaneous species: Beaver, fruit bat, and hairy-footed hamster
• Cat: Cutaneous form caused by wickerhamii is most common but nasal dermatitis from P. zopfii has been identified (Huth, J Comp Pathol. 2015)
• Horses: One case report of Prototheca spp. together with Pithomyces chartarum has been documented as a rare respiratory pathogen (Schöniger, J Comp Pathol. 2016)
• Prototheca in a captive Pteropus lylei: Associated with granulomatous/histiocytic inflammation in the lymph nodes, spleen, meninges/brain, mesentery, pancreas, heart, skeletal muscle, and kidney

REFERENCES:

1. Cheville NF. Algae, fungi, and other eukaryotes. In: Cheville NF, ed. Ultrastructural Pathology The Comparative Cellular Basis of Diseases. 2nd ed. Ames, IA:  Wiley Blackwell; 2009:573-575.
2. 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.
3. Gelatt KN, Plummer CE. Color Atlas of Veterinary Ophthalmology. 2nd ed. Chichester, UK: John Wiley & Sons, Inc; 2017.359.
4. Huth N, et al. Prototheca zopfii genotype 2-induced nasal dermatitis in a cat. J Comp Pathol. 2015;152:287-290.
5. Labelle P. The Eye. In: Zachary JF, eds. Pathologic Basis of Veterinary Disease. 6th ed. Philadelphia, PA: Elsevier; 2017:1301.
6. Lane LV, Meinkoth JH, Brunker J, et al. Disseminated protothecosis diagnosed by evaluation of CSF in a dog. Vet Clin Pathol. 2012;41(1):147-52.
7. Macedo JTSA, Riet-Correa F, Dantas AFM, Simoes SVD. Cutaneous and nasal protothecosis in a goat. Vet Pathol. 2008;45:352-354.
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. Schöniger S, Roschanski N, Rösler U, et. al. Prototheca species and Pithomyces chartarum as causative agents of rhinitis and/or sinusitis in horses. J Comp Pathol. 2016;155:121-125.
11. Uzal FA, Plattner BL, Hostetter JM. Alimentary system. In: Maxie MG, ed. Jubb, Kennedy and Palmer’s Pathology of Domestic Animals. Vol 2. 6th ed. 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:

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