A case of AA amyloidosis with biliary cystadenoma and cervical thymoma in a striped skunk (Mephitis mephitis)

Abstract

A 6-year and 11-months-old male striped skunk, Mephitis mephitis, showed inappetence and died. Necropsy revealed multiple hepatic masses, an enlarged spleen, wedge-shaped lesions in the kidneys, and a cervical cyst with white nodules. Histopathologically, the hepatic masses were diagnosed as biliary cystadenomas accompanied by chronic inflammation, the cervical cyst was a thymoma, and the splenic and renal lesions were diagnosed as infarctions. Amorphous eosinophilic material was observed in the liver, spleen, and kidney. These proteinaceous deposits were positive for Congo red staining with yellowish to green birefringence under polarized light. These deposits were immunohistochemically positive for amyloid A (AA). This report describes the pathological features of AA amyloidosis, biliary cystadenoma and cervical thymoma in a striped skunk.

Skunks belong to the Mephitidae family and are characterized by anal scent glands [8]. Amyloidosis is a group of diseases characterized by the deposition of fibrillar protein in single or multiple organs, and amyloid A (AA) amyloidosis is the most common systemic amyloidosis in animals [7]. In skunks, two reports described AA amyloidosis: one was idiopathic, and the other was considered a sequela of chronic infections [3, 5]. In this study, we describe the pathological features of AA amyloidosis in a skunk (Mephitis mephitis) with biliary cystadenoma and cervical thymoma.

A 6-year and 11-months-old male striped skunk, which was kept at a zoological facility, showed signs of decreased appetite and died. At necropsy, dark-red masses were observed in multiple lobes of the liver (Fig. 1A). The spleen was markedly enlarged. Yellow-red wedge-shaped lesions were observed in the kidneys. In addition, a cyst (3 cm × 3 cm × 4 cm) was observed in the right cervical region (Fig. 2A). The cyst contained clear liquid and white nodules (5–20 mm in diameter) on its wall (Fig. 2B).

Fig. 1.

Liver, striped skunk. (A) Dark-red masses with omental adhesion in multiple lobes. (B) Biliary cystadenoma. Multiple cysts were lined by a single layer of cuboidal epithelial cells and contained eosinophilic substance. Hematoxylin and eosin (HE). (C) Infiltration of lymphocytes and neutrophils around bile duct and amorphous eosinophilic material in sinusoids. HE. (D) The material showed red color under bright field. Congo red staining. (E) The material exhibited yellow to green birefringence under polarized light. Congo red staining. (F) The material is immunolabeled for amyloid A. Immunohistochemistry for amyloid A.

Fig. 2.

Cervical thymoma, striped skunk. (A) A cystic mass lesion in the right cervical region (arrowhead). (B) White nodules (5 mm to 2 cm in diameter) in the cyst wall (arrowheads). (C) Proliferation of polygonal cells with abundant lymphocytes in white nodules. Hematoxylin and eosin. (D) Tumor cells are immunolabeled for cytokeratin AE1/AE3. Immunohistochemistry for cytokeratin AE1/AE3.

For histological examination, tissue samples were fixed in 10% neutral-buffered formalin and embedded in paraffin. Tissues were sectioned at 3-µm thickness for hematoxylin and eosin (HE) staining. Sections of the liver, spleen, kidneys, and heart were stained with alkaline Congo red. Sections of the cervical cyst, liver, spleen, kidneys, and heart were subjected to immunohistochemistry. The following primary antibodies were used: polyclonal rabbit anti-calcitonin (1:100, DAKO, Tokyo, Japan), polyclonal rabbit anti-thyroglobulin (ready to use, DAKO), monoclonal mouse anti-cytokeratin (ready to use, clone AE1/AE3, DAKO), and monoclonal mouse anti-amyloid A (1:200, clone KM268, Kyowa medics, Tokyo, Japan). Deparaffinized tissue sections were treated with 3% hydrogen peroxide (H₂O₂) in methanol at room temperature for 4 min. For antigen retrieval, the sections were treated at 121°C for 10 min in citrate buffer. The sections were incubated in 4% skim milk-Tris-buffered saline at 37°C for 40 min to prevent nonspecific reactions and subsequently incubated with the primary antibodies at 4°C overnight. Anti-mouse or anti-rabbit Envision horseradish peroxidase-labelled polymer (DAKO) was then applied at 37°C for 40 min. Finally, 3–3′-diaminobenzidine (DAB) was used as the chromogen, followed by counterstaining with Mayer’s hematoxylin. As negative controls, each primary antibody was replaced by an irrelevant antibody. Normal thyroid tissue of skunk was used as positive control except for AA.

Histopathologically, the hepatic masses consisted of multiple cysts lined by a single layer of cuboidal epithelial cells, which contained eosinophilic substances and bile (Fig. 1B). These cells were well-differentiated, and had an elongated nucleus on the basal side. No mitotic figures were observed. Severe hemorrhage and lymphocytic infiltration were observed around the tumor tissue. In the Glisson’s capsule of the non-neoplastic area, bile duct proliferation with severe infiltration of lymphocytes and plasma cells were observed. The liver lesions were diagnosed as biliary cystadenoma and chronic cholangitis. In addition, amorphous eosinophilic material was observed in the hepatic sinusoids (Fig. 1C). These proteinaceous deposits were stained with Congo red and showed a red color under bright field and yellow to green birefringence under polarized light (Fig. 1D and 1E). These deposits were immunohistochemically positive for AA (Fig. 1F). Amyloid deposits were also observed in the spleen, renal glomeruli and inner medulla. Based on these findings, the animal was diagnosed with AA amyloidosis.

Histopathological examination of the cervical cyst revealed proliferation of polygonal cells with abundant lymphocytes (Fig. 2C), and the cyst wall consisted of fibrous connective tissue. The polygonal tumor cells had a round nucleus with a prominent nucleolus. Mitotic figures were absent. In addition, cholesterol clefts, macrophage infiltration, and multinucleated giant cells were observed in and around the tumor lesion. Based on the anatomical location and histopathological findings, thymoma and thyroid adenoma were considered differential diagnoses. Therefore, immunohistochemistry for calcitonin, thyroglobulin, and cytokeratin AE1/AE3 was performed using the positive controls. The tumor cells were immunolabeled for cytokeratin AE1/AE3 (Fig. 2D), but not for calcitonin and thyroglobulin. Thus, the cervical lesion was diagnosed as thymoma.

In other organs, multifocal necrosis and wedge-shaped necrosis were observed in the spleen and kidneys. Focal necrosis of the myocardium with macrophage infiltration and angiogenesis was observed in the heart. These lesions may have resulted from multiorgan infarction. The lung was congested and the alveolar spaces were filled with serous fluid, suggesting pulmonary congestion.

The precursor protein of amyloid in AA amyloidosis is serum AA, an acute-phase protein synthesized in the liver [7]. A common cause of AA amyloidosis is supersaturation of circulating SAA associated with persistent inflammation, and amyloid deposits can be observed in the blood vessel walls and interstitium throughout the body [7]. In the present case, chronic cholangitis, which may have been associated with AA amyloidosis, was observed in the liver. In this case, amyloid deposits were observed in the hepatic sinusoids, spleen, and renal glomeruli, which are commonly affected by AA amyloidosis in animals [7]. Infarction and amyloid deposition were observed in the spleen and kidneys. Amyloid deposition in vascular walls often causes angiopathy, and so infarctions in this case may be associated with AA amyloidosis [7].

Previous reports have described the frequency and histopathological features of neoplastic diseases in skunks, including biliary cystadenoma [9, 11,12,13,14, 16, 17]. In a previous study of biliary cystadenomas in a skunk, multiple cystic masses were observed in the liver, consisting of multiple discrete to coalescing, thin-walled, proteinaceous substance-filled cysts of various sizes and shapes, consistent with the present case [16]. Biliary cystadenomas are rare benign tumors in animals but are frequently reported in domestic cats and are suggested to be associated with a higher prevalence of cholangitis [1]. In the present case, chronic cholangitis may have also been caused by biliary cystadenoma.

Thymoma is a neoplastic lesion composed of thymic epithelium with lymphocyte infiltration [18]. It commonly develops in the anterior mediastinum, but may also develop in the cervical region. Cervical thymoma has been previously reported in goats, degu, dogs, and cats [4, 6, 10, 15]. Degu, belonging to the rodent family, has an active cervical thymus for life, similar to other rodents, mice, and rats [2, 15, 19]. Goats also have cervical thymus and thus cervical thymomas are commonly seen. In contrast, cervical thymus is not commonly observed in dogs and cats, and so cervical thymoma in these species is thought to be derived from ectopic thymic tissue [4, 10]. To our knowledge, there are no reports describing the anatomical location of the thymus in the skunks. Therefore, we could not determine whether the thymoma had developed in the cervical thymus or ectopic thymic tissue in the present case. This is the first report of a thymoma in skunks, which is likely a rare tumor in this animal species.

In the present case, the skunk exhibited a decreased appetite, and its digestive tract contained only a liquid composition at necropsy. This may be caused by obstruction of the esophagus by the cervical thymoma. Inappetence and ventral right deviation of the cervical structures were reported in a canine ectopic thymoma case, and increased respiratory sounds and lateral deviation of the trachea were described in a cat [4, 10]. Although such obstruction may be caused by cervical lesions not limited to thymoma, thymoma should be included in the differential diagnosis of cervical mass in skunks.

This is the first report of biliary cystadenoma with AA amyloidosis in a skunk, although there is a case report of biliary cystadenoma [16]. In the present case, AA amyloidosis was thought to be caused by chronic cholangitis associated with biliary cystadenoma. This report also described a cervical thymoma in a skunk for the first time, which may have caused inappetence in the animal due to esophageal obstruction.

CONFLICT OF INTEREST

The authors have no conflicts of interest with respect to the authorship or the publication of this article.