Adult female opossum (Didelphis virginian a).A Good Samaritan found this opossum on a road side and transported it to a wildlife rehabilitation center. The attending veterinarian examined the opossum and found the following: emaciation and dehydration; an abrasion on the right dorsum; a scab with protruding bone at the tail tip; a bruised and swollen left elbow; and numerous small white nodules in the skin of the muzzle, ears and anus. Radiographs revealed a fracture of the left ulna. The opossum died while under isoflurane anesthesia and the attending veterinarian performed a necropsy.

Gross Description:  

There were numerous 1mm in diameter white nodules in the kidney, adrenal glands, spleen, heart, ovary, and haired skin and subcutis. 

Histopathologic Description:

Kidney: Multifocally, throughout the cortex, there are many round protozoal cysts up to 850-1000 μm in diameter that compress adjacent nephrons. The cysts are composed of densely packed crescentic bradyzoites that are 4 μm long surrounded by a 3-8 μm thick rim of host cell cytoplasm which is enclosed in 20-40 μm thick, hyaline capsule. Multifocally, extending from the cortex to the medulla, the interstitium is disrupted and expanded by aggregates of moderate numbers of lymphocytes admixed with fewer plasma cells, and neutrophils. Multifocally, cortical tubules are dilated with attenuated epithelium and contain proteinaceous fluid admixed with few sloughed cells and cellular debris. There are few tubules lined with swollen vacuolated epithelial cells (degeneration). There is also loss of tubules with replacement by fibrous connective tissue. Multifocally, few glomerular tufts are expanded by an eosinophilic homogeneous material.

Morphologic Diagnosis:  

Kidney: Protozoal cysts, numerous, etiology consistent with Besnoitia spp.

Kidney: Nephritis, interstitial, lymphoplasmacytic and neutrophilic, chronic, multifocal, marked, with membranous glomerulonephritis, and interstitial fibrosis.

Not submitted:
Haired skin, ovary, adrenal gland, spleen, and heart: Protozoal cysts, numerous, etiology consistent with Besnoitia spp.

Lung: Bronchopneumonia, granulomatous, multifocal, marked, with adult nematodes, larvae and eggs, etiology consistent with Capillaria spp. and Didelphostrongylus spp.

Spleen: Amyloidosis, multifocal, marked.


Besnoitia darlingi, opossum

Contributor Comment:  

The role that the Besnoitia parasitism played in the pathology of the kidney is not clear. There are Besnoitia cysts surrounded by inflammatory cells and some without, and there are aggregates of inflammatory cells not associated with cysts. The cysts are predominantly intact. Besnoitia is not typically associated with clinical disease in the opossum, though cases with morbidity have been reported.(1,6) While the cause of the chronic interstitial nephritis is not apparent in the submitted sections, this opossum was likely stressed, immunosuppressed and experiencing chronic antigenic stimulation. The open tail wound, the fractured ulna, the skin abrasion, the bronchopneumonia, and the Besnoita all likely contributed to the chronic antigenic stimulation which resulted in the glomerular changes in the kidney and amyloid deposition in the spleen.

Besnoitia is a protozoal parasite in the phylum Apicomplexa.(3) Besnoitia spp. require two hosts (heteroxenous life cycle).(1,6) The domestic cat has been demonstrated to act as a definitive host for Besnoitia darlingi (species associated with opossums), in which infectious oocysts develop and are shed.(7) Opossums and other species act as intermediate hosts, in which oocysts develop into tissue cysts.(2,7) Opossums become infected by ingestion of oocysts from cats or tissue cysts by consumption of infected tissue from other intermediate hosts.(2,6,7)

JPC Diagnosis:  

1. Kidney: Tubulo-interstitial nephritis, neutrophilic and histiocytic, chronic diffuse, marked with intratubular leptospires.
2. Kidney: Amyloidosis, interstitial, multifocal, mild.
3. Kidney: Apicomplexan cysts, multiple.

Conference Comment:  

Besnoitia spp. protozoan parasites infect a wide variety of both wild and domestic mammalian species. The cysts are most commonly seen in visceral organs, skin, and skeletal muscle. As mentioned above, Besnoitia spp. infection in the opossum generally does not produce clinical disease; however, cases with significant lesions and clinical disease have been reported. Clinical disease is generally associated with immunosuppression, stress, and young age. The precise reason some opossums are afflicted by more severe disease is unclear, but other comorbidities may also play a role. In reported clinical cases, protozoal cysts presenting as white nodules at multiple locations in both eyes and nodules throughout the skin were predominant findings; varying degrees and types of ocular pathology were also observed. White nodules were also reported in the oral cavity, lungs, skeletal muscle and heart, specifically in the myocardium where protozoal cysts, foci of mineralization and inflammation were noted. Mineralized hyperechoic nodules were also reported in the kidneys as well as varying degrees of interstitial nephritis and tubular changes. Foci of mineralization were also described in various other tissues.(1) Another study reported protozoal cysts in other organs such as the liver, spleen, stomach and lung. In that same study, debilitated adult female opossums were the most affected subgroup, the ear was the most frequently reported site for protozoal cysts, and the majority of infections were seen in the summer season.(2)

Histologically, the cysts consist of a single enlarged host cell, within which abundant crescent-shaped bradyzoites are packed into a parasitophorous vacuole, which fills the enlarged host cell. The host cell cytoplasm is present as a thin rim at the margin of the enlarged cell. Elongated host cell nuclei may be seen as an inner cyst membrane. The outer layer of the cyst, which forms the capsule, is seen as a variably thick, hyalinized layer of collagen fibers(2) which stains blue with Massons trichrome stain.(5) Mineralization of cysts may be seen as well as varying degrees of host inflammatory reaction.(2) Besnoitia spp. infections are well documented in other mammalian species, including cattle which may become infected with B. besnoiti. Recently, a new cyst nomenclature has been proposed for that species, due to historical inconsistencies in cyst descriptions consisting of the following: Hypertrophied host cell, enlarge nuclei, intracytoplasmic parasitophorous vacuole which contains bradyzoites, an inner cyst wall that may be vacuolated and an outer cyst wall in better developed cysts., and includes tissue cysts This entire structure is referred to as a tissue cyst. In cattle, the condition occurs in acute, subacute and chronic stages. The acute stage is associated with endothelial infections and resultant vascular damage. In subacute and chronic stages, tissue cysts are seen in mesenchymal host cells and are described in a variety of tissues, including being frequently described in the skin.(5)

In this case, the primary lesions, aside from the cysts, include interstitial nephritis, fibrosis, and both tubular and glomerular damage. The interstitial infiltrate is diverse, ranging from predominantly lymphocytes and plasma cells in some areas to being neutrophil- and eosinophil-rich in others. There is mild multifocal intratubular inflammation with tubular degeneration and necrosis. Glomerular changes include membranoproliferative glomerulonephritis and glomeruli range from essentially normal to obsolescent; periglomerular fibrosis is striking in some areas. Amyloid is present in small amounts multifocally within the medullary interstitium, and during the conference, was confirmed with Congo red staining and green birefringence under polarization. The moderator also discussed the presence of extramedullary myelopoiesis, which is multifocal and extensive in some areas of the cortical interstitium. 

In light of the interstitial nephritis and tubular changes, conference participants considered leptospirosis as a primary differential diagnosis. A Warthin-Starry stain identified low numbers of leptospires in the lumen of renal tubules. The opossum has been documented as a reservoir for multiple leptospirosis serovars. Renal lesions due to leptospirosis vary with virulence of the infecting serovar and stage of infection, but generally consist of varying degrees of tubulointerstitial nephritis and tubular necrosis. Subacute or chronic cases have an increased degree of interstitial inflammation and fibrosis may be extensive.(4)


1. Ellis AE, Mackey E, Moore PA, Divers SJ, et al. Debilitation and mortality associated with besnoitiosis in four Virginia opossums (Didelphis virginiana). J Zoo Wildl Med. 2012; 43:367-374.

2. Elsheikha HM, Mansfield LS, Fitzgerald SD, Saeed MA. Prevalence and tissue distribution of Besnoitia darlingi cysts in the Virginia opossum (Didelphis virginiana) in Michigan. Vet Parasitol. 2003; 115:321-327.

3. Gardiner CH, Fayer R, Dubey JP, Service USAR. An Atlas of Protozoan Parasites in Animal Tissues. U.S. Department of Agriculture, Agricultural Research Service. 1988.

4. Greene CE, Sykes JE, Brown CA, Hartmann K. Leptospirosis. In: Greene CE. Ed. Infectious diseases of the dog and cat. 3rd ed. St. Louis, MO: Saunders Elsevier; 2006:402-415.

5. Langenmayer MC, Gollnick NS, Majzoub-Altweck M, Scharr JC. Naturally acquired bovine Bestoitiosis: Histological and immunohistochemical findings in acute, subacute and chronic disease. Vet Pathol. 2015; 52(3):476-488.

6. Shaw S, Grasperge B, Nevarez J, Reed S. Besnoitia darlingi infection in a Virginia opossum (Didelphis virginiana). J Zoo Wildl Med. 2009; 40: 220-223.

7. Smith DD, Frenkel JK. Besnoitia darlingi (Apicomplexa, Sarcocystidae, Toxo-plasmatinae): transmission between opossums and cats. J Protozool. 1984; 31:584-587.

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2-1. Kidney

2-2 Kidney

2-3. Kidney

2-4. Kidney

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