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Benign peripheral nerve sheath tumor in a wild toco toucan (Ramphastos toco).

Abstract: Peripheral nerve sheath tumors are a heterogeneous group of neoplasms that comprise neurofibromas, schwannomas, neurilemmomas, and perineuromas. In animals, peripheral nerve sheath neoplasms are most commonly diagnosed in dogs and cattle, followed by horses, goats, and cats, but their occurrence is uncommon in birds. An adult, free-living, male toco (common) toucan (Ramphastos toco) was admitted to the zoo animal clinic with weight loss, dehydration, and presence of a soft nodule adhered to the medial portion of the left pectoral muscle. Clinical, cytologic, and computed tomography scan results were indicative of a neoplasm. The toucan died during surgical resection of the mass. Necropsy, histopathologic, and immunohistochemical findings confirmed the diagnosis of benign peripheral nerve sheath tumor. To our knowledge, benign peripheral nerve sheath tumor has not previously been reported in a toucan or any other species in the order Piciformes.

Key words: neoplasm, nodule, CD57, immunohistochemistry, Piciformes, wildlife, bird, avian, toco toucan, common toucan, Ramphastos toco

Clinical Report

A free-living adult male toco (common) toucan (Ramphastos toco) was brought to the Clinic for Zoo Animals at the Sorocaba Zoo (Sorocaba, Brazil) because of lethargy. The bird had been found lying on the edge of the highway 3 days previously. The toucan weighed 415 g with complete plumage, indicating that it was an adult. During clinical examination, it was found to be moderately depressed and dehydrated. A soft nodule was found adhered to the medial portion of the left pectoral muscle (Fig 1).

For diagnostic imaging, the bird was anesthetized with isoflurane delivered by mask for induction and then intubated. To better elucidate the size, positioning, and adhesions and branches of the mass, simple and contrast computed tomography (CT) scans (Shimadzu, SCT 7800 TC, Kyoto, Japan) were performed. The contrast study was performed utilizing intravenous 60% diatrizoate meglumine (2 mL/kg). Results of the scan revealed an oval, homogeneous, bilobed mass, hyperattenuating and extending from the caudal third of the neck to the left pectoral muscle. The mass measured 11.6 x 6.9 cm (Fig 2). On the contrast study, the vessels distributed within the mass were evident and were not found to project into the coelomic cavity.

Clinical treatment was initiated with lactated Ringer's solution with 5% dextrose (10 mL SC q12h) and vitamin B complex (3 mg/kg SC q24h). One week later, the toucan was bright and alert, eating, and gaining weight. Once clinical stability was verified, the toucan was anesthetized as described above, and a fine-needle aspirate of the mass was performed for cytologic examination. Meloxicam (0.2 mg/kg IM) was administered for pain management during the procedure. On aspiration, sample material from the mass appeared viscous and hemorrhagic. Results of cytologic examination revealed scattered spindle cells, with minimal atypical findings and no mitotic figures, admixed in a background of red blood cells, suggestive of mesenchymal benign neoplasm. Based on cytologic results the diagnosis was hemangioma. Two weeks after cytologic analysis, the toucan was premedicated with butorphanol (0.5 mg/kg IM) and anesthetized with isoflurane as described above for surgical excision of the mass. Unfortunately, the bird died because of acute subcutaneous hemorrhage during surgical procedure.

At necropsy, the toucan weighed 515 g and was in a good body condition. On dissection, the mass was firmly adhered to the left pectoral muscle (Fig 3). Internal organs appeared normal. Representative sections of all major organs and the mass were collected, fixed in 10% neutral buffered formalin, and submitted for histopathologic and immunohistochemical analyses (Centre of Pathology, Adolfo Lutz Institute, Sao Paulo, Brazil).

Tissue samples were embedded in paraffin wax and trimmed in 3-[micro]m-thick sections for hematoxylin and eosin, alcian blue, and immunohistochemical staining. Microscopic evaluation of the mass revealed an expansive and well-demarked mass, with a scarce, fusiform population of cells surrounded by an abundant myxoid and vascularized background. Cells were elongated with scant cytoplasm and contained an oval nucleus with vesicular chromatin and conspicuous nucleoli. Discreet pleomorphism and low mitotic index were also noted (Fig 4A). The presence of mucus was confirmed in sections stained with alcian blue. No neoplastic cells were observed in any other tissue evaluated.

Antigen retrieval was done with 10 mM citrate buffer (pH 6.0) in a pressure cooker for 3 minutes at 120[degrees]C. Endogenous peroxidase was blocked with 6% hydrogen peroxide for a minimum of 30 minutes. Primary antibodies, anti-CD57 antigens (NK1, Novocastra, Leica Biosystems, Nussloch, Germany), anti-vimentin (V9, Invitrogen, Life Technologies, Carlsbad, CA, USA), anti-pancytokeratins (AE1/AE3, Dako, Glostrup, Denmark), anti-actin smooth muscle (1A4, Dako), antidesmin (D33, Dako), anti-S100 (polyclonal, Dako), anti-CD34 (QBEnd10, Cell Marque, Rocklin, CA, USA), anti-ki-67 (MIB-1/SP6, Dako), anti-Factor VIII (polyclonal, Dako), anti-macrophage (HAM56, Dako), and anti-CD31 (Jc70, Cell Marque), were diluted in 1% bovine serum albumin 0.1% Na3N on phosphate-buffered saline (pH 7.4), followed by overnight incubation (18 hours) at 4[degrees]C. Signal amplification with Picture Max Kit (Life Technologies) secondary peroxidase short-polymer system was carried out for 30 minutes at 37[degrees]C. After developing with 100 mg of 3,3'-diaminobenzidine (D-5637, Sigma, St. Louis, MO, USA), samples were diluted on phosphate-buffered saline (pH 7.4) for 5 minutes at 37[degrees]C. Then, samples were counterstained with Harris hematoxylin for 30 seconds at room temperature, followed by dehydration and slide mounting with synthetic resin. For reaction control, internal positive controls were used. When internal controls were negative, the antibody was considered without cross-reactivity.

In relation to morphologic characteristics, a panel for sarcoma differentiation was chosen. Most of the antibodies applied did not exhibit cross-reactivity with tissues (Table 1). Based on morphologic findings and the results of positive immunostaining for CD57 (NK1) (Fig 4B) and vimentin (V9) (Fig 4C) and negative staining for pancytokeratins (AE1/AE3), smooth muscle actin (1A4), and desmin (D33), a benign peripheral nerve sheath tumor (PNST) was diagnosed in this toucan.


In this toucan, the clinical, tomographic, histologic, and immunohistochemical findings supported the diagnosis of a benign PNST. To our knowledge, this is the first report of a benign PNST in a piciform bird.

Peripheral nerve sheath tumors are a heterogeneous group of neoplasms that comprise neurofibromas, schwannomas, neurilemmomas, and perineuromas. (1) Histologically, each peripheral nerve consists of axons, Schwann cells that form myelin sheaths around some axons, endoneurium, perineurium, and epineurium. (2) Variations of histologic features and differences among species promote controversies regarding the nomenclature of nerve sheath tumors. Thus, malignant or benign PNST is the most commonly used term for animals. (3) In animal species, peripheral nerve sheath neoplasms are most commonly diagnosed in dogs and cattle. (1) In the latter, such neoplasms are typically found as benign masses along the intercostal and epicardial nerves. (1) In birds, the occurrence of PNSTs is rare, with a few reports including a chicken, a Canada goose (Branta canadensis), a macaw (Ara chloroptera), and a golden eagle (Aquila chrysaetos); there are no reports from the order Piciformes, and PNSTs are considered extremely rare in wild birds. (2,4-6)

On the basis of morphologic features and malignant versus benign assessment, tumors can be subdivided into schwannomas, neurofibromas, and malignant PNSTs. (6,7) In veterinary medicine, these tumors are divided into benign or malignant variants because of their uncertain histiogenesis. In the most recent edition of the WHO International Histological Classification of Tumors of Domestic Animals, benign PNSTs are defined histologically as well-circumscribed dermal or subcutaneous tumors composed of wavy spindle cells arranged in bundles, palisades, and whorls, arising from Schwann cells, perineural fibroblasts, or both. (3) Histologically, malignant PNSTs are characterized by interwoven bundles of small, wavy spindle cells with occasional palisading and whorls. (3) They are usually high-grade tumors, with a high mitotic index, necrosis, and metastatic potential. The present case contained a low mitotic index, and almost no atypical cells, which resulted in a benign diagnosis.

Procedures to diagnose PNSTs include CT scans and radiography to identify a soft tissue mass. (6) On CT scan, a PNST appears as a well-defined mass that is hypodense compared with muscle because of the presence of Schwann cells, neural elements, and adipocytes. (8) Enhanced CT scans usually show little or no contrast enhancement. More than half of neurofibromas remain hypodense after contrast injection. (9) Thus, histologic and immunohistochemical examinations can assist in the completion of the diagnosis. (1)

In this case, the positive reaction for vimentin established the mesenchymal origin of the tumor, which is consistent with findings in previous studies. (2,6,9,10) Positivity for anti-CD57 antibody characterizes central and peripheral nervous system cell antigens, as cited by other authors and as has been applied to the diagnosis of PNSTs in dogs. (11) Positive staining for anti-S100 is often associated with the diagnosis of PNSTs in people and dogs, although some cases lack it. (12,13) In this case, there was no cross-reactivity of antibodies with toucan tissues, despite attempts with different dilutions and times of endogenous peroxidase blockage. On histologic examination, the neoplasm in this toucan revealed a loose background rich in mucinous matrix and large vessels. These characteristics can be found in other types of mesenchymal neoplasms such as myxosarcomas, hemangiosarcomas, and liposarcomas. Since no histologic patterns specific for a PNST were identified, the immunohistochemical findings were critical in the diagnosis in this case. Immunohistochemical and histopathologic evaluation favored the diagnosis of benign PNST in this toucan. Proliferation index, by Ki-67 immunolabeling, was attempted to better distinguish between benign and malignant processes, but it revealed no immunoreactivity. Moreover, the neoplasm exhibited an abundant mucinous background, resembling nerve sheath myxoma, a benign entity described in people, but with no animal counterpart. In dogs, similar tumors are named as myxoid benign PNSTs, distinguished from neurofibromas due to lack of nerve fiber bundles surrounded by neoplastic nerve sheath tissue. (14) This subtype of PNST has a benign behavior in people and dogs, but there are no further classifications of PNSTs in birds.

Neoplasms affecting the nervous system are rarely described in birds. (7) Nerve sheath tumors have been reported in a Canada goose, in which findings resembled those presented in this toucan. (5) The goose had firm subcutaneous nodules that ranged in size from 1.2 x 0.8 x 0.8 cm to 7.6 x 4.5 x 2.8 cm within the breast muscles. In another report, a neurofibroma was described in an adult green-winged macaw that initially presented with a firm submandibular, subcutaneous nodule. (4) Three weeks later, the bird developed a second mass on the dorsal surface of the tongue. Diagnosis was confirmed based on results of histologic examination and peroxidase-antiperoxidase testing. In addition, multiple perineuromas were reported in a 2-year-old male Japanese native chicken that presented with an inability to feed due to torticollis. (2) At necropsy, cylindrical enlargements and yellow discoloration of multiple peripheral nerves were observed. On histologic examination, these lesions consisted of a diffuse proliferation of spindle cells with characteristic onion bulb-like structures around residual axons. On immunohistochemical staining, spindle cells were positive for glucose transporter 1 and negative for S-100 proteins. Several authors indicate that multiple intraneural perineuriomas in chickens may be associated with avian leuckosis virus causing fowl glioma. (10) In one report involving a wild bird, a female subadult golden eagle was found lying on the ground with tetraplegia that progressed to a nonambulatory, spastic tetraparesis within a few days. Postmortem magnetic resonance imaging and necropsy findings confirmed the diagnosis of peripheral nerve sheath neoplasm involving a spinal nerve root. (6)

Immunohistochemical staining results are critical for histogenesis conclusions in some neoplasms, especially in mesenchymal neoplasms. In birds, because of the lack of cross-immunoreactivity for commonly used antibodies, more applied research is needed to standardize alternative antibodies. Although peripheral nerve sheath tumors are uncommon in birds, they should be included in the differential diagnosis in any bird presenting with subcutaneous nodules.

Acknowledgments: We thank all the institutions involved in this case report, including the Sorocaba Zoo; the School of Veterinary and Animal Science, State University of Sao Paulo; Adolfo Lutz Institute; and the Laboratory of Wildlife Comparative Pathology, Department of Pathology, University of Sao Paulo. Special thanks to Dr Chris H. Gardiner for the English editing of this manuscript.


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Marcelo P. N. Carvalho, DVM, MSc, Natalia C. C. A. Fernandes, DVM, MSc, Viviane C. Nemer, DVM, Ramiro N. Dias Neto, DVM, MSc, Rodrigo H. F. Teixeira, DVM, MSc, Bruna S. Miranda, DVM, Maria J. Mamprim, DVM, MSc, PhD, Jose L. Catao-Dias, DVM, MSc, PhD, and Rodrigo A. Ressio, MSc, BSc

From the Laboratory of Wildlife Comparative Pathology, Department of Pathology, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Avenida Orlando Marques de Paiva, 87, Cidade Universitaria, Sao Paulo, SP, Brazil, 05508-270 (Carvalho, Catao-Dias); Adolfo Lutz Institute, Centre of Pathology, Department of Pathological Anatomy, Avenida Dr. Arnaldo, 355, Cerqueira Cesar, Sao Paulo, Brazil, 01246-902 (Fernandes, Ressio); Bionicao Veterinary Clinics, Rua Escolastica Rosa de Almeida, 221, Vila Carvalho, Sorocaba, SP, Brazil. 18060-110 (Nemer); Department of Veterinary Surgery and Anesthesiology, School of Veterinary and Animal Science, State University of Sao Paulo, Distrito de Rubiao Junior, s/n, Caixa Postal 560, Botucatu, SP. Brazil, 18618-970 (Dias Neto); Sorocaba Zoo, Rua Teodoro Kaisel, 883, Vila Hortencia, Sorocaba, SP, Brazil, 18020-268 (Teixeira, Miranda); and Department of Animal Reproduction and Veterinary Radiology, School of Veterinary Medicine and Animal Science--University Estadual Paulista (UNESP), Distrito de Rubiao Junior, s/n, Caixa Postal 560, Botucatu, SP, Brazil, 18618-970 (Mamprim).

Table 1. Immunohistochemical results for antigen tests using
various antibodies in tissue samples from a subcutaneous nodule
in a wild adult male toco toucan.

Antibody used          Target cells                Dilution   Method

Anti-CD31              Endothelial cells                50    HRP (b)
Anti-CD34              Endothelial cells               500    HRP
Anti-Ki67              Proliferative cells (G1,        100    HRP
                         G2, S, and M phases)
Anti-SlOO              Human SI00 protein           10 000    HRP
Anti-desmin            Muscle cells                   2000    HRP
Anti-Factor VIII       Endothelial cells               800    HRP
Anti-macrophage        Macrophages                     400    HRP
Anti-pancytokeratins   Epithelial cells               1000    HRP
Anti-smooth muscle     Smooth muscle cells            1000    HRP
Anti-vimentin          Cells of mesenchymal            800    HRP
Anti-CD57              Neuroendocrine origin           200    HRP
                         cells and Schwann cells

Antibody used          Clone        Result (a)

Anti-CD31              Jc70         No cross-immunoreactivity
Anti-CD34              QBEnd10      No cross-immunoreactivity
Anti-Ki67              MIB-1/SP6    No cross-immunoreactivity
Anti-SlOO              Polyclonal   No cross-immunoreactivity
Anti-desmin            D33          Negative
Anti-Factor VIII       Polyclonal   No cross-immunoreactivity
Anti-macrophage        HAM56        No cross-immunoreactivity
Anti-pancytokeratins   AE1/AE3      Negative
Anti-smooth muscle     1A4          Negative
Anti-vimentin          V9           Positive (+++)
Anti-CD57              NK1          Positive (++)

(a) Results: ++ indicates moderately
positive; +++, strongly positive.

(b) HRP indicates peroxidase polymer system detection
(Picture Max Kit, Life Technologies, Carlsbad, CA, USA).
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Author:Carvalho, Marcelo P.N.; Fernandes, Natalia C.C.A.; Nemer, Viviane C.; Neto, Ramiro N. Dias; Teixeira
Publication:Journal of Avian Medicine and Surgery
Article Type:Clinical report
Date:Sep 1, 2016
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