Signalment:  

This was a 2 years of age, male, Collie dog (Canis familiaris) that weighed 55 poundsThis dog was reported to have inappetence and neurologic signs for 3 weeks duration. The neurologic signs included inability to stand, nystagmus, torticollis, and fine tremors when moving the head

Gross Description:  

(Per submitting veterinarian): The lungs had a diffusely gritty consistency. The other reported gross findings were considered to likely be insignificant: the kidneys were slightly softer than normal, the brainstem was slightly softer than normal, and the valve leaflets of the heart were slightly roughened.

Histopathologic Description:

In sections of brain and brainstem, the meninges were thickened by multiple locally extensive infiltrates of mononuclear cells-�-�which were predominately plasma cells, occasional foci of dense fibrin, scattered cellular debris, and numerous round to oval and angular, colorless and refractile organisms measuring 5-15 micrometers. The organisms had a thin, distinct, poorly staining cell wall and contained a nucleus. There were morulae with 2-6 internal round daughter cells measuring 3-8 micrometers and containing 1 nucleus (Fig. 1-1). The organisms were free, as well as phagocytized by macrophages. These organisms were PAS positive (Fig. 1-2) and consistent with Prototheca sp. Variably sized multifocal aggregates of these organisms and identical nonsuppurative inflammatory reactions and necrosis were occasionally within the neuropil and extending into the neuropil from the meninges. 

Sections of pancreas (not submitted) contained multifocal to coalescing areas of necrosis with predominate histiocytes and occasional plasma cells, lymphocytes and neutrophils. Round to oval and angular, 5-15 microns, colorless, refractile organisms were phagocytized in macrophages and free within these necrogranulomatous aggregates.

Sections of lung (not submitted) contained multifocal areas of alveolar collapse and interstitial thickening with neutrophils, macrophages, lymphocytes and plasma cells. 

Morphologic Diagnosis:  

1. Brain: Severe, locally extensive and multifocal, granulomatous meningoencephalitis with intralesional organisms consistent with Prototheca sp.

(Following tissues not included in submitted histologic sections) 2. Pancreas: Moderate to severe, multifocal to coalescing, necrogranulomatous pancreatitis with intralesional organisms consistent with Prototheca sp. 3. Lung: Moderate, multifocal, pyogranulomatous interstitial pneumonia.

Condition:  

Prototheca sp.

Contributor Comment:  

Prototheca is a saprophytic achlorophyllic algae related to the green algae Chlorella. Prototheca thrives in moist environments, and is commonly contracted from lakes and environments with organic debris. The two species associated with disease are Prototheca wickerhamii and Prototheca zopfii. In animals, protothecosis most commonly develops as a systemic infection with or without cutaneous involvement. The organism often has a predilection for the brain and the eyes with resulting neurologic signs or blindness being a common clinical presentation.² Infection usually develops in immunocompromised patients, and Collie dogs are over-represented in reported cases of natural infections. It is believed that with disseminated protothecosis, the organism typically infects the gastrointestinal tract with rapid spread to other organs.³

Prototheca sp. are round, oval, or angular cells that are 8-20 micrometers in diameter, have a refractile wall and contain granular cytoplasm. The organism reproduces by endosporulation and can be seen histologically as morula of 2-20 daughter cells within a single organism. The morula ruptures and releases the individual daughter cells. The cell wall of Prototheca stains poorly with hematoxylin-and-eosin, but stains strongly positive to stains for carbohydrate, such as PAS, Gridley, Bauer, and GMS. 

Prototheca sp. resembles Chlorella sp., but can be distinguished by PAS-positive starch granules in the cytoplasm of Chlorella. In fresh smears of lesions that contain Chlorella, the organisms are green due to the presence of chlorophyll.²

JPC Diagnosis:  

Brain, cerebrum and cerebellum: Meningoencephalitis, granulomatous, multifocal, moderate, with algae, etiology consistent with Prototheca sp., Collie dog (Canis familiaris), canine.

Conference Comment:  

Prototheca sp. are saprophytic organisms that are ubiquitous in the environment, and have been identified on 4 continents.³ They grow in moist environments including sewage, animal feces, slime flux of trees, soil, standing and flowing water, and food.^3,4

Infection is thought to occur through traumatic inoculation, ingestion, or wound contamination. The immune status of an individual appears to play a role in acquiring the infection as well as lesion extent and distribution, although infections have been found in both immunocompetent and immunosuppressed patients.⁴

Clinical manifestation of protothecosis in mammals is to a certain extent species specific. In cattle, mastitis due to P. zopfii is the most common presentation of protothecosis in cattle^1,4 with occasional spread into adjacent lymph nodes, while protothecosis in the dog, also primarily due to P. zopfii⁴, is principally a systemic disease with multi-organ involvement and a predilection for the eyes and brain.^2,3 Protothecosis in cats, due to P. wickerhamii, localizes in the skin, and may be successfully treated by wide surgical excision.^3,4

In dogs the most consistent presenting clinical sign of systemic protothecosis is hemorrhagic colitis.⁴ In fact, the colon and the rectum appear to be primary sites of replication even without clinical evidence of colitis.⁴ A proposed pathogenesis includes initial colonization of the colonic mucosa following ingestion of large numbers of infective organisms, followed by penetration of the gut wall and systemic spread via blood vessels and lymphatics.⁴

Table extracted from Stenner et al.⁴

-	Shape	Sporangia	Sporangiospores
Prototheca zopfii	oval or cylindrical	14-25 um	up to 20
Prototheca wickerhamii	round	7-13 um	up to 50

References:

1. da Costa EO, Ribeiro MG, Ribeiro AR, Rocha NS, de Nardi Junior G: Diagnosis of clinical bovine mastitis by fine needle aspiration followed by staining and scanning electron microscopy in a Prototheca zopfii outbreak. Mycopathologia 158:81-85, 2004
2. Jones T, Hunt R, King N: Diseases caused by fungi. In: Veterinary Pathology, 6th ed., pp. 534-535. Williams & Wilkins, Baltimore, MD, 1997
3. Schultze AE, Ring RD, Morgan RV, Patton CS: Clinical, cytologic and histopathologic manifestations of protothecosis in two dogs. Vet Ophthalmol 1:239-243, 1998
4. Stenner VJ, Mackay B, King T, Barrs VR, Irwin P, Abraham L, Swift N, Langer N, Bernays M, Hampson E, Martin P, Krockenberger MB, Bosward K, Latter M, Malik R: Protothecosis in 17 Australian dogs and a review of the canine literature. Med Mycol 45:249-266, 2007

A virtual slide is not available for this case.

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(Fig 1-1) Cerebellum, meninges

(Fig 1-2) Cerebellum, meninges

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