History
A 14-year-old, 86 kg, domestic huacaya alpaca mare, was referred to the Clinic for Ruminants, Vetsuisse-Faculty, University of Bern, Switzerland for acute dyspnea and inspiratory noise. The mare had not shown any previous respiratory symptoms and had always a good appetite. The referring veterinarian did not initiate any treatment prior to referral.
Clinical findings
Upon clinical examination, the alpaca was bright, alert and in good general condition. Body condition was excellent (BW 86 kg; normal range 55–90 kg) with a body condition score (BCS) of 3 out of 5. Rectal temperature was 37.6 °C (range 37.5–38.9 °C), heart rate 80 beats per minute (range 60–80 bpm) and respiratory rate 28 respiration per minute (range 10–30 rpm). The mare showed mild dyspnea with bilateral dilated nostrils and inspiratory stridor. There was no nasal discharge nor spontaneous or provoked cough. Auscultation of the lung fields revealed bilateral normal sounds, whereas tracheal auscultation revealed a high-pitched stridor. Mucosal membranes were pink and moist and oxygen saturation (Datex Ohmeda Compact s/5 iMM – pulse oxymetry, GE Healthcare, USA) was 95%. Complete blood cell count and blood chemistry were performed and all values were within normal limits.
Diagnostic imaging examinations
Resting endoscopic examination (Silver Scope®, Karl Storz Endoskope, Germany; sheath 7.9 mm, length 140 cm) of the upper airway revealed mild epiglottitis characterized by mucosal edema and reddening, and epiglottic retroversion where the epiglottis retroverted in the opening of the glottis during inspiration and returned to its normal position with each expiration (Fig. 1). Nasal passages and pharynx were considered within normal limits. The trachea could not be examined endoscopically due to the epiglottic retroversion.
Right-to-left lateral radiographic examination (Vertix Vet X-ray system, Siemens, Germany; CR-IR 342, Fuji Photo Film, Japan with FCR Fuji IP Cassette type CC 35.4 × 43 cm; standing animal with slightly extended neck) of the neck showed a wide-based soft tissue mass in the dorsal tracheal wall at the level of the 5th cervical vertebra (C5) (Fig. 2). It markedly decreased the height of the air-filled tracheal lumen and mildly deviated the cervical fascies dorsally. A mild dorsal deviation of the ventral tracheal wall was visible at the same level. The ventrodorsal projection did not provide additional information.
Ultrasound examination (Selfius UF-890AG, Fukuda Denshi, Japan; 7.5 MHz linear probe) revealed the presence of intraluminal tracheal and left juxtatracheal homogeneous hypoechoic masses at the level of the caudal third of the neck (Fig. 3). The extraluminal tracheal mass was in contact with the esophagus. Nevertheless, the motility of this latter was considered normal and not affected by possible adhesions with the mass.
The diagnosis of a tracheal mass was made and a computer tomographic (CT) examination (Brilliance 16, Philips, The Netherlands) was performed under general anesthesia to evaluate the tracheal mass and the surrounding soft tissues in more detail. The CT examination confirmed the presence of an ovoid homogeneous soft tissue mass (LWH: 25.5 × 23 × 14.5 mm), originating from the left dorsal tracheal wall and maximally occluding +/− 90% tracheal lumen at the caudal aspect of the 4th cervical vertebra (C4) (Fig. 4a & b). The inner tracheal lining was separated from the tracheal rings cranial and caudal to this mass. Left to the trachea and dorsal to the esophagus was a second large mildly heterogeneous soft tissue attenuating mass (LWH: 21.5 × 17.5 × 20.5 mm), which focally indented the left tracheal wall and displaced the esophagus ventrolaterally (Fig. 4a & c). This second mass showed border effacement with the trachea and esophagus, but no direct connection between both masses was observed. A small soft tissue nodule (< 1 cm) was identified left ventral to the trachea, approximately 4 cm cranial to the punctum maximum of the intraluminal tracheal mass, likely representing a cervical lymph node. The left superficial cervical lymph node was moderately enlarged (33 × 9 × 2 mm), but still elongated (short axis diameter to long axis diameter ratio = 0.27), flat and with a clearly visible hilus; there were multiple mineralizations present in the normal sized contralateral lymph node (12 × 5 × 4 mm). The submandibular and medial retropharyngeal lymph nodes were symmetrical and within normal size limits.
Cytological findings
Based on physical examination and diagnostic imaging the main differential diagnosis was neoplastic disease. However, a granuloma could not be excluded. A fine needle aspiration (FNA) of the extraluminal mass was therefore performed under ultrasound guidance. Smears were moderately cellular and contained predominantly medium to occasionally large lymphocytes, with a round to mildly indented nucleus (approximately 10 to 15 μm in diameter), coarsely stippled chromatin, occasionally 1–2 small, variably prominent nucleoli, and low to moderate amounts of mid to deeply basophilic cytoplasm (Fig. 5). Scattered mitotic figures were observed.
A diagnosis of lymphoma was therefore issued. Due to the poor prognosis, the owner requested euthanasia of the animal. The animal was euthanized with an intravenous injection of pentobarbital (150 mg/kg IV).
Necropsy findings
The postmortem examination revealed a 2 cm in diameter light pink, round, firm mass firmly attached to the left dorsal tracheal serosa, approximately 30 cm caudal to the larynx (Fig. 6b). Immediately cranial, in the lumen of the trachea, another 2 cm in diameter mass was firmly attached to the mucosa of the dorsal wall of the trachea and displayed a similar morphology as the previously described (Fig. 6a). The esophagus was displaced to the left at the level of the extra luminal mass, but no adhesions were found (Fig. 6b). With the exception of the presence of few mineralized nodules in the liver, the remaining internal organs including the lung and the cervical lymph nodes were macroscopically unremarkable.
Histopathological and immunohistochemical findings (Fig. 7)
Samples of the tracheal masses and from the main internal organs were fixed in 10% neutral buffered formalin for histological examination. The samples were routinely processed, paraffin wax embedded, stained with hematoxylin and eosin (H&E), cut in 3 μm sections and observed in standard light microscopy.
Histologically, the intraluminal tracheal mass consisted of a highly cellular, well demarcated, unencapsulated and infiltrative growing proliferation of neoplastic round cells within the lamina propria, which obstructed the tracheal lumen almost completely and whose surface was focal- extensive ulcerated. The neoplastic cells grew in sheets on a scant amount of highly vascularized fibrous stroma, displayed a small amount of eosinophilic, homogeneous cytoplasm, a round nucleus measuring 1–2 times the length of an erythrocyte with finely stippled chromatin and up to 4 round, basophilic nucleoli (Fig. 7a). The anisocytosis and the anisokaryosis were moderate to high, and there were 14 mitotic figures in ten 400x high-power-fields. In addition, the mass displayed extensive necrotic areas, as well as a multifocal, moderate infiltration with non-neoplastic lymphocytes. The extraluminal tracheal mass displayed similar histological characteristics as the intraluminal mass (Fig. 7d). In addition, a mild suppurative bronchopneumonia, which was most likely due to an impairment of the mucociliary apparatus of the conducting system due to the presence of the intraluminal tracheal mass, and a mild, multifocal chronic granulomatous and fibrosing cholangiohepatitis with multifocal mineralization, which was most likely due to a parasitic infection, could be detected histologically. No further pathological changes were observed histologically in the remaining analyzed internal organs.
Immunohistochemical stains for CD20 (1:200 dilution of Lab Vision™ anti-CD20 rabbit polyclonal antibody, Thermo Fisher Scientific, USA) and CD3 (1:100 dilution of clone LN10 anti-CD3 antibody, Leica Biosystems, Germany) were then performed in order to further characterize these neoplastic lymphocytes. The spleen and a mediastinal lymph node from an alpaca that died due to cachexia and tissue sections from the intraluminal tracheal mass without primary antibody addition were used as positive and negative controls, respectively. The neoplastic lymphocytes in both masses showed a strong positive membranous staining in the CD20 stain (Fig. 7b and e). Occasional CD3 positive lymphocytes displaying both membranous and cytoplasmic staining (Fig. 7c and f), which most likely corresponded to the reactive lymphocytic infiltrates observed in the H&E staining, were also observed. Diagnosis of a B-cell tracheal lymphoma was therefore confirmed.