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Meningeal Granular Cell Tumour in a Green Tree Python (Morelia viridis)
J. Comp. Path. 2020, Vol. 174, 54e57
Available online at www.sciencedirect.com
ScienceDirect www.elsevier.com/locate/jcpa
DISEASE IN WILDLIFE OR EXOTIC SPECIES
Meningeal Granular Cell Tumour in a Green Tree Python (Morelia viridis) D. K. Finnegan*, A. N. Cartoceti†, A. M. Hauck* and E. E. B. LaDouceur* * Veterinary Pathology, Joint Pathology Center, 606 Stephen Sitter Ave, Silver Spring, Maryland and † Smithsonian National Zoo, 3001 Connecticut Ave NW, Washington DC, USA
Summary Granular cell tumours (GCTs) are uncommon neoplasms mostly reported in man, dogs and horses. The origin of GCT is thought to be Schwann cells, with the associated characteristics of neural crest morphology. Neoplastic cells often demonstrate positive immunoreactivity for S100, LC3, vimentin and p62. They are also periodic acideSchiff (PAS) positive and diastase resistant. A female green tree python (Morelia viridis) was presented for severe constipation and hyporexia of 4 month’s duration and, despite treatment, died the next day. A 4.8 3.4 mm intracalvarial GCT was identified, compressing the overlying cerebrum without invasion. Neoplastic cells were immunoreactive to S100 and had brightly eosinophilic cytoplasmic granules that were PAS positive and diastase resistant. Electron microscopy revealed numerous cytoplasmic lysosomes in neoplastic cells. GCTs are reported rarely in non-mammalian species with three reports in birds. This represents the first report of a GCT in a reptile. Ó 2019 Elsevier Ltd. All rights reserved. Keywords: granular cell tumour; green tree python; immunohistochemistry; transmission electron microscopy
Comparative studies suggest that the occurrence of neoplasia in reptiles is similar to that in mammals (Effron et al., 1977). Neoplasia is relatively common in most reptile Orders. Generally, crocodilians and chelonians have a lower reported prevalence of neoplasia than snakes and lizards. Neoplasia of the nervous system is reported rarely in reptiles (Garner et al., 2004). All reported cases were of glial origin except for a single case of a ‘meningeal sarcoma’ (Scott and Beattie, 1927; Craig et al., 2005; Schmidt and Reavill, 2015; Keller et al., 2016; Page-Karjian et al., 2017). Granular cell tumours (GCTs) are uncommon in all animal species. Most GCTs manifest as oral neoplasms in people, dogs and cats (Munday et al., 2017), cerebral meningeal neoplasms in dogs, pituitary gland neoplasms in people (Higgins et al., 2017) and pulmonary neoplasia in people and horses Correspondence to: E. E. B. LaDouceur (e-mail: elise.ladouceur@gmail. com). 0021-9975/$ - see front matter https://doi.org/10.1016/j.jcpa.2019.10.190
(Wilson, 2017). GCTs are thought to arise from the neural crest and are composed of sheets of uniform round to polygonal cells with eosinophilic granules that likely represent autophagosomes or autophagolysosomes. The Schwann cell is considered to be the origin of human GCTs, with recent evidence that canine GCT shares a similar origin. Cells from many canine GCTs have markers of cancer stem cells, can dedifferentiate in culture and redifferentiate in tissue again. GCTs most often demonstrate positive immunoreactivity to S100, LC3, vimentin and p62. Additionally, GCT cells are periodic acideSchiff (PAS) positive and diastase resistant. The majority of these neoplasms are benign, but malignant GCT has also been diagnosed (Suzuki et al., 2015). A 10-year-old female green tree python (Morelia viridis) was presented for severe constipation and hyporexia of 4 month’s duration. Three months prior to case presentation, the snake was examined for suspected neurological deficits (i.e. difficulty righting Ó 2019 Elsevier Ltd. All rights reserved.
Granular Cell Tumour in a Tree Python
and random striking). On clinical presentation, multiple, 1e3 cm diameter masses were palpated in the caudal one-third of the coelom; on radiographs, these masses had mineral opacity and were consistent with eggs. The snake was treated for constipation with a warm water and lubricant enema (30 ml of 50% warm water and 50% lubricant administered via red rubber intermittent catheter into the cloaca), as well as subcutaneous fluids, meloxicam and ceftiofur (both from Zoetis, Parsippany, New Jersey, USA). No significant abnormalities were noted on routine complete blood count and serum biochemical analysis. Despite treatment, the snake was found dead the following morning. At necropsy examination, the snake weighed 897.7 g and was in good body condition with wellfleshed musculature and abundant fat bodies. Gross diagnoses included multifocal myocardial red discolouration with mild epicardial oedema and pericardial effusion; coelomic effusion; segmental colonic mural oedema and multifocal mucosal haemorrhage; and multiple foci of tanegrey discolouration in the liver and lungs. Tissue samples were fixed in 10% neutral buffered formalin, processed routinely and embedded in paraffin wax. Sections (5 mm) were stained with haematoxylin and eosin (HE). Specific tissues (including the entire head) were decalcified in 5% tricholoracetic acid solution (Fisher Scientific, Hampton, New Hampshire, USA) for 3e7 days prior to trimming. An intracranial neoplasm was identified in transverse histological sections of the head. A large (4.8 3.4 mm), well-demarcated, non-infiltrative, densely cellular neoplasm arose from the ventral meninges and markedly compressed the overlying cerebrum and midbrain (Fig. 1). The neoplasm was surrounded by a thin capsule and was composed of sheets of round to polygonal cells supported by scant, delicate, fibrovascular stroma (Fig. 2). Neoplastic cells ranged markedly in size (20e70 mm in diameter) with abundant, brightly eosinophilic, granular cytoplasm. Nuclei had minimal anisokaryosis, ranged from 4 to 5 mm in diameter, and were frequently eccentric to rarely peripheralized. Chromatin was stippled and 1e2 small nucleoli were visible. Additional histological diagnoses (unrelated to the meningeal neoplasm) included ulcerative colitis with transmural haemorrhage (presumed secondary to constipation); mild to moderate heterophilic, segmental enteritis; intravascular bacterial thrombi and epicardial haemorrhage; mild heterophilic pneumonia; multifocal vacuolar hepatopathy (lipid type); intracytoplasmic hepatocyte pigment (presumed haemosiderin); pleural granuloma with acid-fast bacteria
55
Fig. 1. Cross section of the head. A large (4.8 3.4 mm), welldemarcated GCT (asterisk) compresses the overlying cerebrum (arrow) without invasion. HE. Bar, 2 mm.
and hair shafts; and mild heterophilic serositis of the oviduct. Immunohistochemistry (IHC) for S100 and ubiquitin was performed (Walter Reed National Military Medical Center Laboratory, Bethesda, Maryland, USA) on 4 mm sections of the neoplasm. Adjacent cerebral tissue was used as an internal control. Primary antibody applied to each section was anti-S100 (Dako, Carpinteria, California, USA; rabbit polyclonal antiserum at 1 in 200 dilution) and antiubiquitin (Invitrogen, Carlsbad, California, USA; rabbit polyclonal antiserum at 1 in 100 dilution). These antibodies were validated in mammalian, not reptile, species. Reptile-validated S100 and ubiquitin antibodies were not available. Neoplastic cells and control tissues had strong, diffuse, cytoplasmic
Fig. 2. Neoplastic cells have abundant, brightly eosinophilic, granular cytoplasm, a single eccentric to peripheralized small nucleus with finely stippled chromatin, and 1e2 small nucleoli. HE. Bar, 50 mm.
56
D.K. Finnegan et al.
immunoreactivity to S100. Control tissues and the neoplasm were negative for immunoreactivity to ubiquitin; this antibody was considered noncontributory due to lack of positivity in the positive control. PAS and diastase treatment were performed on sections of the neoplasm and on similarly prepared sections of canine liver from the Joint Pathology Center (Silver Spring, Maryland, USA) used for controls. Cytoplasmic granules in neoplastic cells were PAS positive and diastase resistant (Fig. 3). Transmission electron microscopy (TEM) was performed on a sample of the neoplasm removed from the wax block. The sample was dewaxed with changes of xylene, hydrated through a series of graded ethanols and post-fixed in 2% glutaraldehyde. Tissue was prepared for processing and embedded in epoxy resin medium. Thin sections (900 angstr€ oms) were stained with uranyl acetate and lead citrate and examined in a JEOL JEM1400 Electron Microscope (JEOL, Peabody, Massachusetts, USA) and the Gatan Digital Micrograph (Gatan, Pleasantown, California, USA). TEM revealed the neoplastic cell cytoplasm to be expanded by numerous moderately electron-dense lysosomes that contained occasional small vesicles and phospholipid membrane fragments (Fig. 4). These findings are diagnostic for a meningeal GCT. GCTs are reported rarely in non-mammalian species with only three additional reports identified, all of which were in birds (Supplementary Table 1) (Patnaik, 1993; Quist et al., 1999; Hernandez et al., 2012). GCT has not been reported, to the authors’ knowledge, in any amphibians or fish (primary literature and reviews of neoplasia in amphibians and fish were consulted) (Groff, 2004; Stacy and Parker, 2004). All birds with GCTs were psittacines (cockatiel [Nymphicus hollandicus], Puerto Rican
Fig. 3. Neoplastic cells have abundant, bright pink cytoplasmic granules that are PAS positive and diastase resistant, consistent with a GCT. Bar, 50 mm.
Fig. 4. A granular cell nucleus is surrounded by numerous, cytoplasmic, moderately electron-dense lysosomes measuring 0.6e1.2 mm in diameter, and containing occasional small vesicles and phospholipid membrane fragments. The nuclear envelope is highlighted with an arrowhead and the nucleolus with an asterisk. The cellular membrane (arrow) marks the edge of the cytoplasm/cell. The inset is a higher magnification of the lysosomes. TEM. Bar, 2 mm.
Amazon parrot [Amazon vittata ] and Australian parakeet [Melopsittacus undulatus]); two GCTs were in the skin and one in the periocular tissues. In all cases of non-mammalian GCT (including the present case), the diagnosis was presumptive based on histology and was subsequently confirmed with PAS/diastase histochemistry. In three cases (two birds and the present case), the diagnosis was further confirmed with TEM, which consistently revealed abundant cytoplasmic lysosomes. All cases had additional stains and/or IHC (see Supplementary Table 1). Clinical follow up was available in two cases. The Puerto Rican Amazon parrot had no regrowth of the GCT 1 year following surgical removal. The Australian parakeet had incomplete excision (incisional biopsy) of the GCT, which regrew, but was not overtly invasive or associated with any appreciable clinical signs at 7 months after biopsy. Due to the low number of GCTs reported in non-mammalian species, conclusions about biological behaviour are tenuous. However, no evidence of malignancy has been reported in the two avian cases with clinical follow up. In the present case, there were multiple clinical signs that may have been caused by the GCT, specifically hyporexia and an episode of difficulty righting. In mammals, appetite is controlled by the hypothalamus, and destruction of the feeding centre in the lateral hypothalamic area may result in hypo- or anorexia (de Lahunta and Glass, 2009). In reptiles (similar to mammals), the hypothalamus is located at the base of the diencephalon, which was in close approximation and compressed by the GCT in this case. Mammalian and reptilian hypothalami share a similar function, and it is possible that the GCT in this snake compressed the hypothalamus and caused hyporexia (Naumann et al., 2015).
Granular Cell Tumour in a Tree Python
The cause of GCT in this case is unknown. Most cases of neoplasia in reptiles are believed to occur spontaneously, especially in geriatric animals (Frye, 1994). This snake was considered slightly older than middleaged with lifespans of green tree pythons estimated at approximately 15 years (Wilson et al., 2006). Neoplasia of the nervous system is reported rarely in snakes, possibly because the brain is difficult to access in small or venomous specimens. In all small to medium-sized reptiles, the authors recommend examination of the brain in situ via preparing decalcified sections of the head. This allows for identification of unexpected lesions of the nervous system that could easily be overlooked, as these stoic animals may have few to no nervous signs despite substantial nervous lesions, as in this case.
Acknowledgments The authors thank institutions and individuals for contributing case material to this study, including the Animal Care Sciences and Wildlife Health Sciences staff at the Smithsonian National Zoological Park. We thank E. Perezrosaria and A. Cummings Tasker for preparation of material for TEM and the histology staff at the Joint Pathology Center and Walter Reed National Military Medical Center for preparation of slides, IHC and special stains, especially W. McNeil, S. Tamer, S. Mcnair, K. Gathers, A. Brown and A. Cherilus. The views expressed in this abstract are those of the authors and do not reflect the official policy of the Department of the Army/Navy/Air Force, Department of Defense or US Government.
Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.jcpa.2019.10.190.
References Craig LE, Wolf JC, Ramsay EC (2005) Spinal cord glioma in a ridge-nosed rattlesnake (Crotalus willardi). Journal of Zoo and Wildlife Medicine, 36, 313e315. de Lahunta A, Glass E (2009) Diencephalon. In: Veterinary Neuroanatomy and Clinical Neurology, 3rd Edit., Saunders Elsevier, St. Louis, pp. 483e486. Effron M, Griner L, Benirschke K (1977) Nature and rate of neoplasia found in captive wild mammals, birds, and reptiles at necropsy. Journal of the National Cancer Institute, 59, 185e198. Frye FL (1994) Diagnosis and surgical treatment of reptilian neoplasms with a compilation of cases 1966e1993. In Vivo, 8, 885e892. Garner MM, Hernandez-Divers SM, Raymond JT (2004) Reptile neoplasia: a retrospective study of case submissions to a specialty diagnostic service. Veterinary Clinics of North America: Exotic Animals, 7, 653e671.
57
Groff JM (2004) Neoplasia in fishes. Veterinary Clinics of North America: Exotic Animals, 7, 705e756. Hernandez V, Carrera E, Mendez A, Morales JC, Morales E et al. (2012) Histopathologic, immunohistochemical and ultrastructural features of a granular cell tumour in an Australian parakeet (Melopsittacus undulatus). Avian Pathology, 41, 437e440. Higgins RJ, Bollen AW, Dickinson PJ, Siso-Llonch S (2017) Tumors of the nervous system. In: Tumors of Domestic Animals, D Meuten, Ed., John Wiley & Sons, Ames, pp. 870e872. Keller KA, Sanchez-Migallon Guzman D, Sanders C, Tong N, Mohr FC et al. (2016) Clinical and pathological findings in a red-tailed boa constrictor (Boa constrictor constrictor) with a primary neural neoplasm within the diencephalon and mesencephalon. Journal of Herpetological Medicine and Surgery, 26, 85e89. Munday JS, Lohr CV, Kiupel M (2017) Tumors of the alimentary tract. In: Tumors of Domestic Animals, D Meuten, Ed., John Wiley & Sons, Ames, pp. 511e512. Naumann RK, Ondracek JM, Reiter S, Shein-Idelson M, Tosches MA et al. (2015) The reptilian brain. Current Biology, 25, R317eR321. Page-Karjian A, Hahne M, Leach K, Murphy H, Lock B et al. (2017) Neoplasia in snakes at Zoo Atlanta during 1992e2012. Journal of Zoo and Wildlife Medicine, 48, 521e524. Patnaik AK (1993) Histologic and immunohistochemical studies of granular cell tumors in seven dogs, three cats, one horse, and one bird. Veterinary Pathology, 30, 176e185. Quist CF, Latimer KS, Goldade SL, Rivera A, Dein FJ (1999) Granular cell tumour in an endangered Puerto Rican Amazon parrot (Amazon vittata). Avian Pathology, 28, 345e348. Schmidt RE, Reavill D (2015) Diseases of the head of amphibians and reptiles. Proceedings of the Association of Reptilian and Amphibian Veterinarians Conference, San Antonio, 571e578. Scott HH, Beattie J (1927) Neoplasm on a porose crocodile. Journal of Pathology & Bacteriology, 30, 61e66. Stacy BA, Parker JM (2004) Amphibian oncology. Veterinary Clinics of North America: Exotic Animals, 7, 673e695. Suzuki A, Uchida K, Harada T, Nibe K, Yamashita M et al. (2015) The origin and role of autophagy in the formation of cytoplasmic granules in canine lingual granular cell tumors. Veterinary Pathology, 52, 456e464. Wilson D, Heinsohn R, Wood J (2006) Life-history traits and ontogenetic colour change in an arboreal tropical python, Morelia viridis. Journal of Zoology, 270, 399e407. Wilson DW (2017) Tumors of the respiratory tract. In: Tumors of Domestic Animals, D Meuten, Ed., John Wiley & Sons, Ames, pp. 495e496.
September 16th, 2019 ½ Received, Accepted, October 21st, 2019
Available online at www.sciencedirect.com
ScienceDirect www.elsevier.com/locate/jcpa
DISEASE IN WILDLIFE OR EXOTIC SPECIES
Meningeal Granular Cell Tumour in a Green Tree Python (Morelia viridis) D. K. Finnegan*, A. N. Cartoceti†, A. M. Hauck* and E. E. B. LaDouceur* * Veterinary Pathology, Joint Pathology Center, 606 Stephen Sitter Ave, Silver Spring, Maryland and † Smithsonian National Zoo, 3001 Connecticut Ave NW, Washington DC, USA
Summary Granular cell tumours (GCTs) are uncommon neoplasms mostly reported in man, dogs and horses. The origin of GCT is thought to be Schwann cells, with the associated characteristics of neural crest morphology. Neoplastic cells often demonstrate positive immunoreactivity for S100, LC3, vimentin and p62. They are also periodic acideSchiff (PAS) positive and diastase resistant. A female green tree python (Morelia viridis) was presented for severe constipation and hyporexia of 4 month’s duration and, despite treatment, died the next day. A 4.8 3.4 mm intracalvarial GCT was identified, compressing the overlying cerebrum without invasion. Neoplastic cells were immunoreactive to S100 and had brightly eosinophilic cytoplasmic granules that were PAS positive and diastase resistant. Electron microscopy revealed numerous cytoplasmic lysosomes in neoplastic cells. GCTs are reported rarely in non-mammalian species with three reports in birds. This represents the first report of a GCT in a reptile. Ó 2019 Elsevier Ltd. All rights reserved. Keywords: granular cell tumour; green tree python; immunohistochemistry; transmission electron microscopy
Comparative studies suggest that the occurrence of neoplasia in reptiles is similar to that in mammals (Effron et al., 1977). Neoplasia is relatively common in most reptile Orders. Generally, crocodilians and chelonians have a lower reported prevalence of neoplasia than snakes and lizards. Neoplasia of the nervous system is reported rarely in reptiles (Garner et al., 2004). All reported cases were of glial origin except for a single case of a ‘meningeal sarcoma’ (Scott and Beattie, 1927; Craig et al., 2005; Schmidt and Reavill, 2015; Keller et al., 2016; Page-Karjian et al., 2017). Granular cell tumours (GCTs) are uncommon in all animal species. Most GCTs manifest as oral neoplasms in people, dogs and cats (Munday et al., 2017), cerebral meningeal neoplasms in dogs, pituitary gland neoplasms in people (Higgins et al., 2017) and pulmonary neoplasia in people and horses Correspondence to: E. E. B. LaDouceur (e-mail: elise.ladouceur@gmail. com). 0021-9975/$ - see front matter https://doi.org/10.1016/j.jcpa.2019.10.190
(Wilson, 2017). GCTs are thought to arise from the neural crest and are composed of sheets of uniform round to polygonal cells with eosinophilic granules that likely represent autophagosomes or autophagolysosomes. The Schwann cell is considered to be the origin of human GCTs, with recent evidence that canine GCT shares a similar origin. Cells from many canine GCTs have markers of cancer stem cells, can dedifferentiate in culture and redifferentiate in tissue again. GCTs most often demonstrate positive immunoreactivity to S100, LC3, vimentin and p62. Additionally, GCT cells are periodic acideSchiff (PAS) positive and diastase resistant. The majority of these neoplasms are benign, but malignant GCT has also been diagnosed (Suzuki et al., 2015). A 10-year-old female green tree python (Morelia viridis) was presented for severe constipation and hyporexia of 4 month’s duration. Three months prior to case presentation, the snake was examined for suspected neurological deficits (i.e. difficulty righting Ó 2019 Elsevier Ltd. All rights reserved.
Granular Cell Tumour in a Tree Python
and random striking). On clinical presentation, multiple, 1e3 cm diameter masses were palpated in the caudal one-third of the coelom; on radiographs, these masses had mineral opacity and were consistent with eggs. The snake was treated for constipation with a warm water and lubricant enema (30 ml of 50% warm water and 50% lubricant administered via red rubber intermittent catheter into the cloaca), as well as subcutaneous fluids, meloxicam and ceftiofur (both from Zoetis, Parsippany, New Jersey, USA). No significant abnormalities were noted on routine complete blood count and serum biochemical analysis. Despite treatment, the snake was found dead the following morning. At necropsy examination, the snake weighed 897.7 g and was in good body condition with wellfleshed musculature and abundant fat bodies. Gross diagnoses included multifocal myocardial red discolouration with mild epicardial oedema and pericardial effusion; coelomic effusion; segmental colonic mural oedema and multifocal mucosal haemorrhage; and multiple foci of tanegrey discolouration in the liver and lungs. Tissue samples were fixed in 10% neutral buffered formalin, processed routinely and embedded in paraffin wax. Sections (5 mm) were stained with haematoxylin and eosin (HE). Specific tissues (including the entire head) were decalcified in 5% tricholoracetic acid solution (Fisher Scientific, Hampton, New Hampshire, USA) for 3e7 days prior to trimming. An intracranial neoplasm was identified in transverse histological sections of the head. A large (4.8 3.4 mm), well-demarcated, non-infiltrative, densely cellular neoplasm arose from the ventral meninges and markedly compressed the overlying cerebrum and midbrain (Fig. 1). The neoplasm was surrounded by a thin capsule and was composed of sheets of round to polygonal cells supported by scant, delicate, fibrovascular stroma (Fig. 2). Neoplastic cells ranged markedly in size (20e70 mm in diameter) with abundant, brightly eosinophilic, granular cytoplasm. Nuclei had minimal anisokaryosis, ranged from 4 to 5 mm in diameter, and were frequently eccentric to rarely peripheralized. Chromatin was stippled and 1e2 small nucleoli were visible. Additional histological diagnoses (unrelated to the meningeal neoplasm) included ulcerative colitis with transmural haemorrhage (presumed secondary to constipation); mild to moderate heterophilic, segmental enteritis; intravascular bacterial thrombi and epicardial haemorrhage; mild heterophilic pneumonia; multifocal vacuolar hepatopathy (lipid type); intracytoplasmic hepatocyte pigment (presumed haemosiderin); pleural granuloma with acid-fast bacteria
55
Fig. 1. Cross section of the head. A large (4.8 3.4 mm), welldemarcated GCT (asterisk) compresses the overlying cerebrum (arrow) without invasion. HE. Bar, 2 mm.
and hair shafts; and mild heterophilic serositis of the oviduct. Immunohistochemistry (IHC) for S100 and ubiquitin was performed (Walter Reed National Military Medical Center Laboratory, Bethesda, Maryland, USA) on 4 mm sections of the neoplasm. Adjacent cerebral tissue was used as an internal control. Primary antibody applied to each section was anti-S100 (Dako, Carpinteria, California, USA; rabbit polyclonal antiserum at 1 in 200 dilution) and antiubiquitin (Invitrogen, Carlsbad, California, USA; rabbit polyclonal antiserum at 1 in 100 dilution). These antibodies were validated in mammalian, not reptile, species. Reptile-validated S100 and ubiquitin antibodies were not available. Neoplastic cells and control tissues had strong, diffuse, cytoplasmic
Fig. 2. Neoplastic cells have abundant, brightly eosinophilic, granular cytoplasm, a single eccentric to peripheralized small nucleus with finely stippled chromatin, and 1e2 small nucleoli. HE. Bar, 50 mm.
56
D.K. Finnegan et al.
immunoreactivity to S100. Control tissues and the neoplasm were negative for immunoreactivity to ubiquitin; this antibody was considered noncontributory due to lack of positivity in the positive control. PAS and diastase treatment were performed on sections of the neoplasm and on similarly prepared sections of canine liver from the Joint Pathology Center (Silver Spring, Maryland, USA) used for controls. Cytoplasmic granules in neoplastic cells were PAS positive and diastase resistant (Fig. 3). Transmission electron microscopy (TEM) was performed on a sample of the neoplasm removed from the wax block. The sample was dewaxed with changes of xylene, hydrated through a series of graded ethanols and post-fixed in 2% glutaraldehyde. Tissue was prepared for processing and embedded in epoxy resin medium. Thin sections (900 angstr€ oms) were stained with uranyl acetate and lead citrate and examined in a JEOL JEM1400 Electron Microscope (JEOL, Peabody, Massachusetts, USA) and the Gatan Digital Micrograph (Gatan, Pleasantown, California, USA). TEM revealed the neoplastic cell cytoplasm to be expanded by numerous moderately electron-dense lysosomes that contained occasional small vesicles and phospholipid membrane fragments (Fig. 4). These findings are diagnostic for a meningeal GCT. GCTs are reported rarely in non-mammalian species with only three additional reports identified, all of which were in birds (Supplementary Table 1) (Patnaik, 1993; Quist et al., 1999; Hernandez et al., 2012). GCT has not been reported, to the authors’ knowledge, in any amphibians or fish (primary literature and reviews of neoplasia in amphibians and fish were consulted) (Groff, 2004; Stacy and Parker, 2004). All birds with GCTs were psittacines (cockatiel [Nymphicus hollandicus], Puerto Rican
Fig. 3. Neoplastic cells have abundant, bright pink cytoplasmic granules that are PAS positive and diastase resistant, consistent with a GCT. Bar, 50 mm.
Fig. 4. A granular cell nucleus is surrounded by numerous, cytoplasmic, moderately electron-dense lysosomes measuring 0.6e1.2 mm in diameter, and containing occasional small vesicles and phospholipid membrane fragments. The nuclear envelope is highlighted with an arrowhead and the nucleolus with an asterisk. The cellular membrane (arrow) marks the edge of the cytoplasm/cell. The inset is a higher magnification of the lysosomes. TEM. Bar, 2 mm.
Amazon parrot [Amazon vittata ] and Australian parakeet [Melopsittacus undulatus]); two GCTs were in the skin and one in the periocular tissues. In all cases of non-mammalian GCT (including the present case), the diagnosis was presumptive based on histology and was subsequently confirmed with PAS/diastase histochemistry. In three cases (two birds and the present case), the diagnosis was further confirmed with TEM, which consistently revealed abundant cytoplasmic lysosomes. All cases had additional stains and/or IHC (see Supplementary Table 1). Clinical follow up was available in two cases. The Puerto Rican Amazon parrot had no regrowth of the GCT 1 year following surgical removal. The Australian parakeet had incomplete excision (incisional biopsy) of the GCT, which regrew, but was not overtly invasive or associated with any appreciable clinical signs at 7 months after biopsy. Due to the low number of GCTs reported in non-mammalian species, conclusions about biological behaviour are tenuous. However, no evidence of malignancy has been reported in the two avian cases with clinical follow up. In the present case, there were multiple clinical signs that may have been caused by the GCT, specifically hyporexia and an episode of difficulty righting. In mammals, appetite is controlled by the hypothalamus, and destruction of the feeding centre in the lateral hypothalamic area may result in hypo- or anorexia (de Lahunta and Glass, 2009). In reptiles (similar to mammals), the hypothalamus is located at the base of the diencephalon, which was in close approximation and compressed by the GCT in this case. Mammalian and reptilian hypothalami share a similar function, and it is possible that the GCT in this snake compressed the hypothalamus and caused hyporexia (Naumann et al., 2015).
Granular Cell Tumour in a Tree Python
The cause of GCT in this case is unknown. Most cases of neoplasia in reptiles are believed to occur spontaneously, especially in geriatric animals (Frye, 1994). This snake was considered slightly older than middleaged with lifespans of green tree pythons estimated at approximately 15 years (Wilson et al., 2006). Neoplasia of the nervous system is reported rarely in snakes, possibly because the brain is difficult to access in small or venomous specimens. In all small to medium-sized reptiles, the authors recommend examination of the brain in situ via preparing decalcified sections of the head. This allows for identification of unexpected lesions of the nervous system that could easily be overlooked, as these stoic animals may have few to no nervous signs despite substantial nervous lesions, as in this case.
Acknowledgments The authors thank institutions and individuals for contributing case material to this study, including the Animal Care Sciences and Wildlife Health Sciences staff at the Smithsonian National Zoological Park. We thank E. Perezrosaria and A. Cummings Tasker for preparation of material for TEM and the histology staff at the Joint Pathology Center and Walter Reed National Military Medical Center for preparation of slides, IHC and special stains, especially W. McNeil, S. Tamer, S. Mcnair, K. Gathers, A. Brown and A. Cherilus. The views expressed in this abstract are those of the authors and do not reflect the official policy of the Department of the Army/Navy/Air Force, Department of Defense or US Government.
Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.jcpa.2019.10.190.
References Craig LE, Wolf JC, Ramsay EC (2005) Spinal cord glioma in a ridge-nosed rattlesnake (Crotalus willardi). Journal of Zoo and Wildlife Medicine, 36, 313e315. de Lahunta A, Glass E (2009) Diencephalon. In: Veterinary Neuroanatomy and Clinical Neurology, 3rd Edit., Saunders Elsevier, St. Louis, pp. 483e486. Effron M, Griner L, Benirschke K (1977) Nature and rate of neoplasia found in captive wild mammals, birds, and reptiles at necropsy. Journal of the National Cancer Institute, 59, 185e198. Frye FL (1994) Diagnosis and surgical treatment of reptilian neoplasms with a compilation of cases 1966e1993. In Vivo, 8, 885e892. Garner MM, Hernandez-Divers SM, Raymond JT (2004) Reptile neoplasia: a retrospective study of case submissions to a specialty diagnostic service. Veterinary Clinics of North America: Exotic Animals, 7, 653e671.
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September 16th, 2019 ½ Received, Accepted, October 21st, 2019