Metastatic Cholangiocarcinoma in a Bearded Dragon (Pogona vitticeps)

Abstract A 6.5-year-old female bearded dragon (Pogona vitticeps) was presented with a swollen right pelvic limb. A tissue core biopsy from the swollen area was performed and a presumptive histopathological diagnosis of adenocarcinoma was made. This diagnosis was confirmed after limb amputation. Two months after amputation a sudden deterioration in the overall health of the patient occurred. Ultrasound examination of the coelomic cavity revealed hypoechoic lesions in the liver. The patient was euthanized and submitted for necropsy which revealed a severely enlarged liver with multiple coalescing yellowish nodules. Cholangiocarcinoma of the liver with metastases to the spleen, left mesovarium and right pelvic limb was diagnosed after histopathological examination.


INTRODUCTION
Diagnoses of neoplastic diseases are increasing amongst reptiles due to their growing popularity as pet animals [1].
Differential diagnoses of swelling in reptiles are bacterial granulomas, neoplastic disorders, parasitic cysts, fungal granulomas, epidermal cysts, hematomas, aneurysms and gout or pseudogout [19,20]. The fi nding of a mass on a reptile´s body does not immediately mean that the lesion is neoplastic. The appropriate management of the swelling without histological or cytological confi rmation is not possible [21]. Therefore, it is very useful to biopsy such lesions and perform further examinations.
The aim of this case report is to describe a metastatic cholangiocarcinoma of the liver in a bearded dragon. During the review of available literature regarding neoplasia in bearded dragons, the authors did not fi nd any reported case of cholangiocarcinoma with metastases to the spleen, mesovarium and pelvic limb.

CASE PRESENTATION
A 6.5-year-old female bearded dragon (Pogona vitticeps), weighing 430 g, was presented by zookeepers to the veterinary clinic in Ljubljana Zoo due to swelling of the right pelvic limb. The bearded dragon was owned by the Zoo and was kept in a terrarium with sandy substrate together with four other females. The food mainly consisted of insects such as crickets, grasshoppers, fl our worms and a minimal herbivorous diet, mainly lettuce.
Clinical examination revealed a deteriorated nutritional status with a body condition score of 2/5. The body orifi ces showed no discharge and the animal's breathing was calm. A marked swelling of the right shin with skin ulcerations was the main fi nding (Fig. 1). The swelling was approximately 2.5 cm in diameter and the skin ulceration  about 4 mm in diameter. The skin and dermal derivatives on other parts of the body were without abnormalities. The swollen area was stiff, slightly sensitive to palpation and extended from the stifl e to the tarsal joint. X-ray examination (HiRay Plus X-ray Machine, Eickemeyer Veterinary Equipment Ltd., Sunbury-on-Thames, UK) of the affected limb showed pronounced swelling of the soft tissues, whereas no radiological abnormalities were detected on the bone structures. Due to the assumption of an infl ammatory process, anti-infl ammatory and analgesic therapy with meloxicam (0.5 mg/kg) (Meloxidyl 5 mg/ml, Ceva Santé Animale, Libourne, France) (subcutaneously, q 24 h) and antibiotic therapy with marbofl oxacin (10 mg/kg) (Marbocyl 10%, Vetochinol, Cedex, France) (subcutaneously, q 48 h) was administered empirically [22].
Slight improvement of the clinical status was noticed after 2 weeks of therapy. This improvement was refl ected in a reduction of the skin ulceration. Blood from the coccygeal vein was collected for biochemical examination and the results were within normal limits. Anti-infl ammatory and antibiotic therapy was prolonged, and the skin ulcerations were treated daily with antimycotic terbinafi ne (Lamisil 1% Cream, Novartis s.r.o., Prague, Czech Republic). After an additional 2 weeks the clinical status did not improve, therefore biopsy samples of the soft tissue from the affected area were obtained for histopathological examination under local anaesthesia. Tramadol (2 mg/kg) (Tramal 50 mg/ml, Stada Arzneimittel AG, Bad Vilbel, Germany) [22] and meloxicam (0.5 mg/kg) were used as premedication and 4 mg/kg of lidocaine (Lidocain 2%, Egis Pharmaceuticals PLC, Budapest, Hungary) was administered into the muscles proximal to the stifl e. Two punch biopsies were obtained, fi xed in 10% buffered formalin and sent for histopathology. A presumptive diagnosis of adenocarcinoma of unknown origin was the result of the histopathological examination (Fig. 2a). A control X-ray after biopsy revealed a transverse single-stranded fracture of the fi bula (Fig. 3).
After receiving the histopathological diagnosis of adenocarcinoma, amputation of the affected limb was performed and the limb was sent for histopathological examination. The premedication chosen was the same as that administered when sampling for the histopathological examination -tramadol (2 mg/kg) and meloxicam (0.5 mg/kg). Induction of anaesthesia was performed by intravenous administration of propofol (5 mg/kg) (Propofol 2% MCT/LCT Fresenius, Fresenius Kabi Deutschland GmbH, Bad Homburg, Germany) into the ventral coccygeal vein. Subsequently, the patient was intubated by means of an intravenous catheter (Vasofi x Safety 16G, B. Braun Melsungen, Melsungen, Germany) and the maintenance of anaesthesia was carried out with a mixture of oxygen (2 L/min) and 3% of isofl urane (Aerrane, Baxter S.A., Lessines, Belgium). The limb was amputated at the stifl e and the stump was stitched in two layers with a horizontal mattress stitch (PDS 4-0; Johnson & Johnson Medical N.V., Belgium). A bolus of Ringer solution (20 ml/kg) (B. Braun Melsungen, Melsungen, Germany) together with Duphalyte (20 ml/kg) (Pfi zer Olot SLU, Vall de Bianya, Spain) in a total volume of 10 ml was administered subcutaneously after the operation.
Recovery from anaesthesia was without complications. Prokinetic medication with metoclopramide (2 mg/kg) (Vomend 5mg/ml; Hyperdrug Pharmaceuticals Ltd, Middleton-in-Teesdale, UK) was administered subcutaneously after the operation and artifi cial feeding of the patient by oesophageal tube was conducted for 5 days (PD Hill's Urgent Care Canine/Feline a/d, Hill's Pet Nutrition Inc., USA). Application of meloxicam and marbofl oxacin was prolonged in the above-mentioned doses for one week after the operation. Histopathological examination of the amputated pelvic limb confi rmed the initial histopathological diagnosis of adenocarcinoma. The tumour infi ltrated the dermis, striated muscles and also bone tissue, where it extended into the medullary cavity (Fig.  2c).
Sudden deterioration of the overall health condition occurred two months after amputation of the affected limb. The patient was apathetic and stopped eating. Ultrasound examination of the coelomic cavity was performed by Mindray M7 (Mindray DS USA Inc., Mahwah, USA) with an 8 MHz microconvex transducer. Signifi cant enlargement of the liver with presence of multiple cystic anechoic structures with clear distal signal amplifi cation was observed (Fig. 4) and liver neoplasia was the presumed diagnosis. Blood was obtained for haematological and control biochemical examination. All biochemical and haematological parameters were within normal range. Due to poor clinical status and the ultrasonographic fi nding, the animal was euthanized. A combination of propofol (5 ml pro toto) and T61 (1.5 ml pro toto) (Intervet International BV, Boxmeer, The Netherlands) was administered into the ventral coccygeal vein. Successful euthanasia was verifi ed by acoustic oscillometric examination of the heart. Subsequently, the animal was referred for necropsy. A markedly enlarged liver with multiple coalescing yellowish nodules measuring 2 to 14 mm in diameter, which were multifocally cystic, was the major fi nding at necropsy. The affected liver measured 10 x 9 x 2 cm and occupied one third of the entire coelomic cavity. The gallbladder was signifi cantly dilated and contained 5 ml of green, translucent bile (Fig. 5). There was 30 ml of slightly cloudy, reddish free fl uid in the coelomic cavity. A single, slightly protruding, white-yellow nodule measuring 5 mm in diameter was found on the left part of the fat body and on the surface of the left lung lobe. The right ovary was not present in the coelomic cavity, whereas the left ovary contained several small follicles.
All organs from the coelomic cavity and the brain were collected for histopathology. Most of the liver parenchyma was replaced by variably sized tumorous tubular structures, predominantly composed of a single layer of columnar cells, which expressed moderate anisocytosis, had a moderate amount of lightly basophilic cytoplasm and a round to oval, moderately anisokaryotic nucleus with one or two nucleoli, or the nucleolus was not visible (Fig. 2b). The mitotic index was 4 mitoses per 10 high power fi elds; rare mitoses were bizarre. Multifocally, there were groups of neoplastic cells in the spleen and fat tissue around the left ovary and the fallopian tube which were morphologically identical to the neoplastic cells in the liver. The macroscopically observed nodule on the left part of the fat body proved to be liponecrosis and the nodule on the left lung lobe was determined to be a chronic granuloma. Sewing thread with concurrent granulomatous infl ammation was present in the fat under the left ovary. The kidneys showed moderate cholemic nephrosis. In other organs of the coelomic cavity and the brain no histopathological changes were found.

DISCUSSION
Many swellings in reptiles are caused by abscesses [23]. Because of this fact, antibiotic and anti-infl ammatory medication was the empirical fi rst choice of therapy in this case. Fungal infection is another frequent cause of limb swelling, including the fi ngers. Surface ulceration in swelling caused by fungal infections is often observed [24,25]. Such ulceration was also observed in the described case, but there was no improvement after daily antimycotic administration.
Due to a wide range of different diagnoses of swelling on reptiles´ bodies biopsy is useful for externally palpable masses [21]. The best method for determining the diagnosis of neoplastic disease is tissue biopsy [1,21]. Reavill (2004) suggests fi neneedle aspiration biopsy of the affected area [26]. We chose to perform punch biopsy under local anaesthesia in our case so as to provide an adequate amount of tissue for histopathological examination.
We encounter many types of neoplasia in reptiles which are already known in mammals [26,27]. Cholangiocarcinoma of the liver was diagnosed after necropsy in this case. Cholangiocarcinoma is a malignant neoplasia that originates from the biliary epithelium. In domestic animals, such as dogs, cats, cattle, sheep and horses, it is less frequent than hepatocellular tumours. In these animals cholangiocarcinomas are known to metastasize to the drainage lymph nodes, lungs and also to the peritoneal serosal surfaces [28].
Only swelling of soft tissues without detectable osteolysis of the bone structures was detected on the primary X-ray. A fi bula fracture, without osteolysis, was evident in the affected area in the repeated X-ray examination after 4 weeks. It is possible that the fracture was caused by frequent manipulation of the limb during treatment or during biopsy (the control X-ray was performed after the punch biopsy).
We suppose that the swelling of the right pelvic limb was a result of metastasis of the primary cholangiocarcinoma of the liver into the limb, because there is no primary glandular tissue in this region from which the tumour could have originated. When the overall clinical condition of the patient deteriorated and ultrasonographic examination of the coelomic cavity revealed a severely enlarged liver, a primary tumour of the liver was highly suspected. Diagnostic imaging methods were not performed initially since the patient had no clinical symptoms except unilateral pelvic limb swelling. On the basis of this experience, the authors recommend a thorough screening examination of the animal's body using imaging methods to determine whether it is a primary or metastatic neoplastic process.