Acta Veterinaria Hungarica 57 (4), pp. 477–484 (2009) DOI: 10.1556/AVet.57.2009.4.2

0236-6290/$ 20.00 © 2009 Akadémiai Kiadó, Budapest

HAEMANGIOMA IN THE OESOPHAGUS OF A RED-EARED SLIDER (TRACHEMYS SCRIPTA ELEGANS)

János GÁL1*, Csaba JAKAB1, Zoltán SZABÓ2, Péter PAZÁR2, Roland PSÁDER2, Florian ROEBER5, Árpád HEGYI3, Kinga Katalin LEFLER3, Balázs FARKAS4 and Míra MÁNDOKI1

1Department of Pathology and Forensic Veterinary Medicine and 2Department and Clinic of Internal Medicine, Faculty of Veterinary Science, Szent István University, István u. 2, H-1078 Budapest, Hungary; 3Department of Fish Culture, Institute of Environmental and

Landscape Management, Faculty of Agriculture and Environmental Sciences, Szent István University, Gödöllő, Hungary; 4European Freshwater Turtle Breeders’

Association, Gyúró, Hungary; 5Faculty of Veterinary Science, The University of Melbourne, Werribee, Victoria 3030, Australia

(Received 13 November 2008; accepted 23 March 2009)

A haemangioma developing in the wall of the oesophagus and protruding into its cavity is reported for the first time from a Red-eared Slider (Trachemys scripta elegans). As the tumour mechanically hampered swallowing, the animal was unable to eat and consequently developed a poor condition. Histopathology of the tumour revealed all characteristics of a haemangioma: the blood-filled blood-vessels having an irregular cross-section were lined with endothelial cells. Claudin-5 immunohistochemical antibodies were employed for characterising the tumour, and this examination confirmed our initial diagnosis of a haemangioma.

Key words: Red-eared Slider (Trachemys scripta elegans), oesophagus, haemangioma, immunohistochemistry

Reptiles, amongst them turtles and tortoises, are getting diagnosed with tumours at an increasing rate (Gál et al., 2002; Garner et al., 2004; Gál et al., 2006; Gál et al., 2007). In a study based on nearly a decade of research the occur-rence of tumours in chelonians had a frequency of 2.7%; while tortoises were af-fected in only 1.4%, the frequency of occurrence in freshwater turtles was as high as 3.2% (Garner et al., 2004). In another study encompassing a much longer period and deriving from 490 cases, the frequency of tumour occurrence was 1.2% (Sykes and Trupkiewicz, 2006). According to Garner et al. (2004), the most frequent type of tumour was fibropapilloma (34.4%), followed by squamous cell carcinoma (10.3%). While they did not mention tumours of blood-vessels in che-

*Corresponding author; E-mail: Gal.Janos@aotk.szie.hu; Phone: 0036 (1) 478-4178; Fax: 0036 (1) 478-4284

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lonians, these authors reported haemangiosarcoma to occur with 1.5% frequency in snakes and 0.6% in lizards, and haemangioma with only 0.3% frequency in snakes and 2.5% in lizards. Other general works also do not mention tumours originating from blood vessels in chelonians (e.g., Mader, 2006).

In the suborder Serpentes, tumours developing in blood vessels were re-ported from the gastrointestinal tract, the liver, the skin as well as the ovary, and the taxa affected included crotalid, boid and colubrid snakes (McArthur et al., 2004; Mader, 2006). In a single case a cysts-like swelling was found in the clo-aca of a Southern Pacific Rattlesnake (Crotalus viridis helleri) (Mader, 2006), and a haemangioendothelioma originating from the liver and paralysing the entire body was observed in a Cook’s Tree Boa (Epicrates cooki) (Frye, 1991; Mader, 2006). Also a Boa constrictor was diagnosed with skin haemangioendothelioma, which caused skin ulcer (Mader, 2006). Its tumour consisted of blood-filled si-nuses (Frye, 1991). Haemangiomas originating from the ovary were diagnosed in two species of crotalid snakes, the Eastern Massasauga (Sistrurus catenatus cate-natus) and the Timber Rattlesnake (Crotalus horridus) (Mader, 2006). In the fam-ily Colubridae a Pacific Gopher Snake (Pituophis melanoleucus catenifer) was diagnosed with liver haemangiosarcoma (Sykes and Trupkiewicz, 2006).

Haemangiomas can be diagnosed by histopathology, but these tumours can easily be mistaken for other types of tissue swelling or vascular malforma-tions. Recently, claudin-5 (tight junction) protein expression has been tested in veterinary oncology for identification of the vascular endothelium of lymph and blood vessels (Jakab et al., 2008). This protein has been introduced as a marker for differentiated endothelial cells, based on immunohistochemical methods.

Materials and methods

Animal

An adult (11 years old) male Red-eared Slider with a plastron length of 17 cm and a mass of 190 g was presented for inspection on 16 December 2007 at the Department and Clinic of Internal Medicine, Faculty of Veterinary Science, Szent István University, Budapest. The turtle had previously been housed in an aquarium with the dimensions 40 × 50 × 25 cm (length × width × height), at a water level of approximately 15 cm. It had access to a flat stone for basking. The water had not specially been heated, so its temperature ranged between 18 and 29 °C, depending on the room temperature. The animal had been fed on a varied diet at every 2–3 days. Besides frozen fish it had been offered smaller water snails, mealworms, rarely pieces of cow liver and chicken meat as well as lettuce. The appetite of the animal decreased by the end of the summer and it started to ac-cept less food. One month prior to our inspection it completely ceased feeding even though it appeared hungry. It took the food in its jaws but was apparently

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unable to swallow it. At the same time the owner recognised a considerable swell-ing at the lower part of the turtle’s neck.

Sample collection and processing

The physical investigation of the animal included a thorough examination of its skin, carapace and plastron as well as all body openings, after which its overturning reflexes were evaluated. This was followed by laterolateral and dorsoventral radiography (60 kV, 0.3 sec, 6.5 mAs ) of the animal’s neck, and af-ter a superficial narcosis with a 5 mg/kg body mass dose of zolazepam–tiletamin combination (Zoletil, Virbac) a video-endoscopic investigation was carried out [light sense 201325 20, Karl Storz (Germany), camera control unit 692360 20, Karl Storz (Germany), videobronchoscope 11900BP, videocard Aver TV GO 007 FM plus] by which the oropharyngeal cavity, the oesophagus and the stom-ach were inspected.

As the physical, radiographic and endoscopic findings pointed to an inoper-able tumour, the owner requested the humane euthanasia of the animal, which was done by administering a 200 mg/kg body mass dose of ketamine (CP-KetaminR) in accordance with the welfare regulations valid in Hungary and the European Union.

The skin on the ventral surface of the neck was cut open in a longitudinal direction, the pathological lesion revealed by radiography was investigated, and a sample was fixed in 4% neutral-buffered formalin for further histological studies. The plastron of the turtle was then removed and the organs of the peritoneal cav-ity were examined.

The sample fixed in 4% neutral-buffered formalin was subsequently em-bedded in paraffin wax and a 4 µm thick section was cut, which was stained with haematoxylin and eosin. Based on the results of haematoxylin-eosin staining fur-ther immunohistochemical examinations were done. The immunohistochemical antibodies used were CD31 (diluted 1:80, JC/70A, mouse anti-human mono-clonal, DAKO) and claudin-5 (diluted 1:100, 4C3C2, mouse anti-human mono-clonal, Zymed Inc., San Francisco, CA, USA – #18-7364). For immunohisto-chemistry 3–4 µm thick sections were cut. The slides for immunohistochemical reactions were deparaffinised in xylene and graded ethanol. The deparaffinised sections were treated with primary antibody CD31 or claudin-5 for 60 min at room temperature after treatment with appropriate antigen retrieval (Target Re-trieval Solution, DAKO, Glostrup, Denmark, pH 6; 800 W microwave oven for 30 min). Immunohistochemical staining was performed using the streptavidin-peroxidase technique. Antigen-bound primary antibody was detected using stan-dard avidin-biotin immunoperoxidase complex (DAKO LSAB2 Kit). The chro-mogen substrate was 3, 3-diaminobenzidine tetrahydrochloride (DAB substrate-chromogen, DAKO, Denmark). Mayer’s haemalaun was used for counterstain-ing. For the immunohistochemical reaction, a negative control with omission of the primary antibody was included.

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Results

During the physical examination the animal appeared agile, active and the overturning reflexes were adequate. Its carapace, plastron and skin were charac-teristic of males of this taxon. The turtle’s body openings were clear and intact. A walnut-sized, dense, flexible lesion was observed in the neck region.

Laterolateral and dorsoventral radiographs of the neck region of the turtle revealed normal head and neck ossification, with no signs of pathologic changes of their anatomy. In the central, lower part of the neck a nearly spherical, homo-geneous shadow cast formed by soft tissue and having a diameter of approxi-mately 5 cm could be observed (Fig. 1).

Endoscopic examination revealed no lesions in the oropharyngeal cavity and the cranial part of the oesophagus. Approximately 6–7 cm from the mouth orifice a soft tissue densely perfused by blood vessels was seen protruding from the wall of the oesophagus and strongly narrowing the lumen. A cloudy, viscous discharge was present here in large amounts. Beyond this lesion the oesophagus had a regular shape, the mucous membrane of the stomach appeared greyish-white, wrinkled, with some clear, viscous material in between the wrinkles (Fig. 2).

By cutting open the skin of the neck in a longitudinal direction, dissection revealed oedema and dark red discoloration in the tissues surrounding the oe-sophagus. After cutting through the oesophagus, an approximately 2–3 cm wide tumour with a halved orange-like surface and a dark red colour was detected around the lumen of the oesophagus (Fig. 1).

Upon opening the peritoneal cavity no adipose tissue was visible in the physiological fat reserves. The stomach and the intestines contained very little viscous content, but they appeared to be normal.

Histologically, the oesophageal mass was sharply defined, but not encap-sulated, and was made up of large, cavernous vascular spaces filled with blood and nucleated erythrocytes, lined by one layer of well-differentiated endothelial cells and separated by fibrous connective tissue. Cellular atypia was lacking, and no mitotic figures were noted. Slit-like vascular channels were occasionally ar-ranged in a radiating pattern around a mature well-differentiated vessel. Some cavernous spaces were distended up to 2 mm in diameter by partially organised thrombi. Thrombosis and endothelial damage were associated with haemorrhage and necrosis of adjacent tissue. Fibrous septa between vascular channels varied in density and cellularity (Fig. 3).

In the tissue mass, the regularly used panendothelial marker, CD31 did not show any positivity either in the tumour or in the normal tissue, but claudin-5 re-acted specifically with differentiated endothelial cells. Intense staining of claudin-5 was detected in the membrane of neoplastic endothelial cells (Fig. 3), proving the presence of a haemangioma. The epithelial cells of the oesophageal mucosa were used as internal control and showed the positive brown membrane staining.

Acta Veterinaria Hungarica 57, 2009

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Fig. 1. Radiogram and macroscopic picture of the neck region of a Red-eared Slider. A: dorsoventral radiogram, B: laterolateral radiogram,

C: perioesophageal oedema, D: tumour in the oesophagus wall

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482 G

ÁL et al.

Fig. 2. Videoendoscopic picture of the oesophagus and the gastric cavity. A: intact oesophagus section, B: tumour developing in and protruding

from the oesophagus wall, C: intact stomach mucous membrane

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Fig. 3. Histological section of the tumour (A: haematoxylin-eosin staining, × 200, B: claudin-5

immunohistochemical reaction, × 400). 1: epithelium lining the oesophagus, 2: connective tissue framework of the tumour, 3: caverns filled with blood and lined with endothelial cells, 4: endothe-

lial cells showing positive reaction for claudin-5 (the membrane is coloured brown, intensive membrane-bound claudin-5 positivity in the oesophageal epithelium as internal control)

Discussion

We have diagnosed a haemangioma arising from the oesophageal wall of a Red-eared Slider, strongly narrowing the cavity of the oesophagus. Both the macro- and microscopic features of the tumour led to the diagnosis of a haeman-gioma. This tumour rendered swallowing difficult, and ultimately hampered it completely. As a consequence, the animal used up its body fat reserves and its condition deteriorated substantially. Physical, radiographic and endoscopic in-vestigations all revealed the presence of large-sized intramural tissue growth that appeared inoperable. Because of this and upon the owner’s request the turtle was euthanised.

According to the literature, several lesions narrowing the lumen of the oe-sophagus may hamper the swallowing of food, e.g. oedema in the neck region, haematoma, inflammatory processes and thyroid gland enlargement (Mader,

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2006). However, no haemangioma originating from the wall of the oesophagus has ever been described in a Red-eared Slider, and we are unaware of any report of either benign or malignant tumours originating from blood vessels in chelonians.

The generally used immunohistochemical marker CD31, which reacts with the differentiated endothelial cells of humans, cats, dogs and horses, did not function. The claudin-5 tight junction protein is more conservative, so it can be used as an endothelial marker in a variety of different species. Given that claudin-5 reacted positively with the differentiated neoplastic endothelial cells of the tu-mour, it appears to be a suitable tool for characterising haemangioma in chelo-nians and possibly other reptiles as well.

References

Frye, F. L. (1991): Biomedical and Surgical Aspects of Captive Reptile Husbandry. Chapter 16. Krieger Publishing Co., Malabar, Florida, USA.

Gál, J., Albert, M. and Dobos-Kovács, M. (2002): Myeloid leukaemia in black head python (Aspid-ites melanocephalus) (in Hungarian, with English abstract). Magyar Állatorvosok Lapja 124, 491–495.

Gál, J., Jakab, Cs., Balogh, B., Tóth, T. and Farkas, B. (2007): First occurrence of periosteal chon-droma (juxtacortical chondroma) in Uromastyx maliensis. Acta Vet. Hung. 55, 327–331.

Gál, J., Szabó, Gy., Jakab, Cs., Géczy, Cs. and Sátorhelyi, T. (2006): Adenocarcinoma with squamous metaplasia in the parathyroid gland of Spur-thighed Tortoise (Testudo graeca) (in Hungarian, with English abstract). Magyar Állatorvosok Lapja 128, 632–637.

Garner, M. M., Hernandez-Divers, S. M. and Raymond, J. T. (2004): Reptile neoplasia: a retro-spective study of case submission to a specialty diagnostic service. Vet. Clin. Exot. Anim. Pract. 7, 653–671.

Jakab, Cs., Halász, J., Kiss, A., Schaff, Zs., Szabára, Á., Rusvai, M. and Kulka, J. (2008): Exami-nation of claudin-5 protein expression of the endothelial cells of lymph vessels in canine mammary glands and mammary gland carcinoma by immunohistochemical methods (in Hungarian, with English abstract). Magyar Állatorvosok Lapja 130, 296–303.

Mader, D. R. (2006): In: Reptile Medicine and Surgery. Chapter 19. Saunders-Elsevier, St. Louis, Missouri.

McArthur, S., Wilkinson, R. and Meyer, J. (2004): Medicine and Surgery of Tortoises and Turtles. Chapter 13. Blackwell Publishing, Ames, Iowa.

Sykes, J. M. and Trupkiewicz, J. G. (2006): Reptile neoplasia at the Philadelphia Zoological Gar-den, 1901–2002. J. Zoo Wildl. Med. 37, 11–19.