Case of the Month - December 2025

Signalment and History

This juvenile, intact male Virginia opossum was admitted to a wildlife rehabilitator in a weakened and underweight state. Distinctly outlined radiolucent foci were detected in the cortical bone of several long bones. Due to the state of the animal and a poor prognosis for recovery, the animal was euthanized on the day of admission.

Gross Findings

The animal was emaciated and moderately dehydrated. White nodules up to 0.2 cm in diameter were present in liver, lung, kidneys (Figure 1 A), skeletal muscle, periosteum (Figure 2A), and the left eye. Faxitron-radiographs of the kidney were characterized by the presence of numerous spherical foci with a radiolucent core that was distinctly outlined by a radiodense rim

Figure 1: (A) Gross image of the kidney demonstrating multifocal to coalescing, variably sized, white nodules expanding the renal parenchyma, mostly confined in the cortex. (B) Radiograph of the same kidney (as in A) highlighting multiple radiolucent foci corresponding to the nodular lesions.

Figure 2: (A) Gross image of a long bone demonstrating a well-demarcated, nodule arising from the periosteum (white arrow). (B) Radiograph of the same long bone highlighting the discrete radiolucent nodule (white arrow) that was observed grossly. Other similar nodules were noticed on radiograph (orange thin arrows) within the periosteum.  

Histopathology

Liver, lung, kidney, bone marrow, brain, eye, stomach (muscularis layer): The affected tissue contains variably distributed protozoal cysts, ranging from solitary to numerous, measuring up to approximately 120 μm in diameter. The cysts are round to oval and delineated by a thick, amphophilic wall (Figure 3B). Within the cysts are numerous, elongated to spindle-shaped (bradyzoites) (Figure 3B) with eosinophilic cytoplasm and a single basophilic nucleus. Often cysts are degenerate and filled with necrotic cellular debris. Occasionally, ruptured cysts (Figure 3C) are observed, associated with granulomatous inflammation, characterized by aggregates of macrophages, multinucleated giant cells, lymphocytes, and fewer plasma cells.

Figure 3: (A) Photomicrograph of a protozoal cyst within the myocardium, consistent with B. darlingi. (B) Higher magnification of the cyst demonstrating a thick cyst wall enclosing numerous crescent-shaped bradyzoites. (C) Granulomatous inflammation in the myocardium surrounding and infiltrating a degenerate cyst, actively phagocytosing cyst remnants. (D) Photomicrograph of a degenerate cyst within the renal parenchyma. The outer lining (capsule) of the cyst is circumferentially calcified while the lumen contains necrotic debris of host cells and bradyzoites. (E) Photograph of a protozoal cyst in the cerebellum with associated granulomatous inflammation.

Ancillary Testing

• Faxitron radiology of long bones and kidneys: Numerous radiolucent foci were present in the kidneys (matching mineralized Besnoitia darlingi cysts, Figure 3C) (Figure 1B). Few radiolucent foci were present in the cortices of long bones (matching Besnoitia darlingi cysts) (Figure 2B).
• Parasitology: Few Eimeria sp. oocysts were observed in the intestinal content.

Morphologic Diagnosis

1. Cerebellum, meningitis, granulomatous, focally extensive, with intralesional degenerate (partially mineralized) protozoal cyst (presumptive Besnoitia sp.)
2. Heart, myocarditis, granulomatous, multifocal, mild with few intralesional degenerate (partially mineralized) protozoal cysts (presumptive Besnoitia sp.)
3. Kidney, nephritis, granulomatous, multifocal, mild with numerous intralesional degenerate (partially mineralized) protozoal cysts (presumptive Besnoitia sp.)
4. Liver, hepatitis, granulomatous, multifocal, mild with few intralesional degenerate (partially mineralized) protozoal cysts (presumptive Besnoitia sp.)

Etiology

Besnoitia darlingi

Comments

Besnoitia spp. are apicomplexan protozoa with a heteroxenous life cycle. Besnoitia darlingi is a Besnoitia sp. that is specific to Virginia opossums. The life cycle of Besnoitia darlingi includes felids—particularly domestic cats and bobcats—as definitive hosts shedding oocysts, while the opossums act as intermediate hosts harboring tissue cysts. Histologically, these cysts represent hypertrophied host cells containing numerous bradyzoites within a parasitophorous vacuole, surrounded by a variably thick capsule. 

One may question whether this animal’s death resulted from disseminated infestation with protozoal cysts, leading to subsequent emaciation and dehydration, or whether the animal was already debilitated and therefore prone to a widespread, overwhelming protozoal infection. This distinction is particularly relevant, as Besnoitia darlingi infection is very common in Virginia opossums and usually regarded as incidental. While some cysts, including a cyst in the cerebellum of the animal, elicited a granulomatous response—particularly when ruptured—most were intact and minimally reactive, consistent with the typically low pathogenicity of this parasite in opossums. However, in the context of stress, immunosuppression, and poor body condition, Besnoitia infections have been associated with more severe and disseminated disease. In this case, the high parasite burden may reflect either opportunistic proliferation in an immunocompromised host or a contributing factor to systemic decline, although distinguishing cause from effect remains challenging.

The remarkable radiographs in this case, particularly of the kidneys and long bones, highlight a notable feature of Besnoitia cysts: when the capsule is mineralized, they can appear as distinctly radiodensely outlined structures in soft tissue while the viable protozoal cysts appear as distinct radiolucent foci in the cortical bone of long bones. The value of the kidney radiograph lies in the demonstration of a severity of the renal involvement, with histopathologic analysis showing that the majority—if not all—of the capsules of the observed cysts were mineralized (Figure 3D), resulting in an astonishing radiographic appearance (Figure 1B).

While not detected in this opossum, it is important to recognize that Virginia opossums (Didelphis virginiana) serve (or are suspected to serve) as definitive host for several host-adapted parasites that typically do not negatively affect the opossums themselves. Notably, opossums serve as the definitive host for Sarcocystis falcatula and are strongly implicated as the definitive host for Sarcocystis neurona. While birds, such as eagles, are usually the intermediate hosts for Sarcocystis falcatula, and can die from the infection when the central nervous system is affected, horses exposed to opossum fecal matter can act as intermediate hosts for Sarcocystis neurona. Horses with Sarcocystis neurona infection can develop severe neurologic signs (“protozoal equine myeloencephalitis”) necessitating euthanasia. These relationships highlight the epidemiologic importance of opossums in the transmission of pathogens across species.

References

1. Ellis, Angela. Debilitation and Mortality Associated with Besnoitiosis. Journal of Zoo and Wildlife Medicine. 2012. 43(2): 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 Aug 14;115(4):321-7. doi: 10.1016/s0304-4017(03)00221-8. PMID: 12944045.
3. Verma, Shiv. K, et al. Bobcats (Lynx rufus) are natural definitive host of Besnoitia darlingi. Veterinary Parasitology. 2017. 248 (2017): 84-89.