Antemortem Diagnosis of Multicentric Lymphoblastic Lymphoma, Lymphoid Leukemia, and Inclusion Body Disease in a Boa Constrictor (Boa constrictor imperator)

Inclusion body disease (IBD) is a fatal disease of snakes, first described in captive boids in 1994 (Schumacher et al., 1994). It generally affects members of the Boidae and Pythonidae families (Carlisle-Nowak et al., 1998; Chang and Jacobson, 2010; Keilwerth et al., 2012; Orós et al., 1998; Schumacher, 2006; Vancraeynest et al., 2006), although it has also been reported in palm vipers (Bothriechis marchi) (Raymond et al., 2001) and an eastern king snake (Lampropeltis getulus) (Jacobson et al., 2001). The disease is characterized by large eosinophilic intracytoplasmic inclusions in neurons and epithelial cells of various organs (Carlisle-Nowak et al., 1998; Chang and Jacobson, 2010; Jacobson et al., 2001; Keilwerth et al., 2012; Orós et al., 1998; Raymond et al., 2001; Schumacher, 2006; Vancraeynest et al., 2006; Wozniak et al., 2000). Recently, arenaviruses have been isolated from snakes suffering from IBD, and are suspected to be the etiological agents of this disease (Bodewes et al., 2013; Hetzel et al., 2013; Stenglein et al., 2012).

Lymphoma, also know as lymphosarcoma, is a malignant hematopoietic neoplasia derived from solid lymphoid organs (thymus, spleen, bone marrow). Neoplastic lymphoid cells tend to invade other internal organs and cause diffuse infiltration. Grossly, these tumors appear as solid whitish-grey masses with frequent areas of necrosis and hemorrhage (Reavill, 2004). About 20 cases of lymphomas have been reported in various reptile species, as summarized in Table 1. In most of these cases, the animals were presented with lethargy, anorexia, and loss of condition, and further investigations usually revealed the presence of a mass and/or edema (Jacobson et al., 1980, 1981; Schultze et al., 1999; Folland et al., 2011). A few cases revealed specific abnormalities upon examination and a specific diagnosis was only determined following necropsy (Orós et al., 2001; Raiti et al., 2002; Gyimesi et al., 2005; Schilliger et al., 2011). Other cases describe subcutaneous tumors (Schultze et al., 1999), diffuse tumors causing secondary cervical edema (Folland et al., 2011), or multiple small whitish nodules on the thymus, thyroid, heart, aortic arches, lungs, spleen, liver, kidney, stomach, or small intestine (Gyimesi et al., 2005; Orós et al., 2001).

The term leukemia is restricted to the neoplastic proliferation of hematopoietic stem cells, i.e., “blasts,” which derive from bone marrow. They can be of lymphoid or myeloid origin and are classified according to cell type and the degree of maturity of the cell (acute or chronic) (Frye and Carney, 1972; Elkan and Cooper, 1976; Machotka, 1984; Garner et al., 2004; Reavill, 2004; Mauldin and Done, 2006). Leukemia generally causes nonspecific signs in both reptiles and mammals, including anorexia, weakness, lethargy, loss of condition, and occasional mild to moderate lymphadenopathy in mammals (Morris and Dobson, 2001).

The purpose of the present case report is to describe, to the best of the authors' knowledge for the first time in a live snake, the diagnosis of concurrent lymphoblastic lymphoma, lymphoid leukemia, and IBD.

A 6-yr-old male boa constrictor (Boa constrictor imperator) was presented for anorexia, lethargy, and postural abnormalities that had developed over several weeks. The owner had also noticed the recent appearance of a coelomic mass in the cervical region. The animal weighed 1.85- kg and was a captive-bred specimen bought from a pet shop at the age of 6 months. Since then it had been kept by the same owner and had never been in contact with another snake. The animal was regularly dewormed and had, according to the owner, never been sick or undergone surgery. The terrarium was optimally set up for a boa constrictor in terms of environmental temperature (28–32°C [82–90°F] daytime and 24–26°C [75–79°F] nighttime) and relative humidity (60–80%) (Rossi, 2006; Russo, 2007). The heating mat was installed so as to provide a temperature gradient across the terrarium. Lighting was provided for 10 h daily by a fluorescent tube without ultraviolet B irradiation. The snake's diet consisted of a live rat presented every 20 days. The terrarium included branches suited for an arboreal lifestyle.

When placed onto the examination table, the animal was lethargic despite a good body condition. Its movements were slow and nonfluid, and the righting reflex was absent when placed into dorsal recumbency. A swelling was observed 20 cm distal to the snout on the ventrum. On palpation, a large (4 cm × 2 cm × 1 cm) firm, nonmobile mass could be palpated inside the coelomic cavity. This mass was distinct from the heart, as visualized by the heart contractions roughly 5–6 cm caudal to it. No other abnormalities were palpated in the coelomic cavity. Examination of the buccal cavity did not reveal any abnormalities; mucous membranes were of normal color and there were no signs of stomatitis or abnormal secretions from the glottis. Cardiac auscultation with the use of a Doppler flow probe (Vet Dopp®, Heska Corporation, Fort Collins, CO) of 8 MHz showed a normal heart rate (30 beats per min at 30°C, 27 beats per min when using the formula of the prediction of heartbeat frequency: hbf = 33.4 [Wt_kg^−0.25]) (Murray, 2006). No wheezes or rales were heard on pulmonary auscultation.

Hematology (complete blood count and cytology) and serum biochemistry analyses were done in order to narrow down the differential list. A blood sample was taken by cardiocentesis (Isaza et al., 2004; Brown, 2010), and the blood was placed in EDTA and lithium heparin tubes. Blood smears were made to screen for IBD. Complete blood count showed anemia (0.33 × 10⁶/μL, range: 1–2.5), severe leukocytosis (102.0 × 10³/μL, range 4–10), lymphocytosis (91.8 × 10³/μL, range 10–60), a low number of heterophils (2.04 × 10³/μL, range 0.8–6.5), and the presence of atypical cells (Rosskopf et al., 1982; Frye, 1991b; Carpenter et al., 2001; Diethelm and Stein, 2006; Sykes and Klaphake, 2008). Blood smear cytology (May Grünwald Giemsa stain [MGG]) revealed hematologic malignancy suggestive of a lymphoid cell origin (lymphoid leukemia). It showed an increased density of atypical medium- to large-sized round cells, showing ovoid nuclei with slightly noncondensed chromatin, and frequent visible nucleoli. Occasional binucleated cells were seen. These atypical round cells had a clear basophilic cytoplasm. Based on the appearance, the atypical cells were consistent with blast cells. Few mitoses were seen. Intracytoplasmic inclusion bodies were found in numerous erythrocytes (Fig. 1A).

Plasma biochemistries revealed hyperphosphatemia (9.3 mg/dL, range 2.6–4.9), hyperproteinaemia (9.1 g/dL, range 4.6–8.0), increased uric acid (6.22 mg/dL, range 1.2– 5.8), a marked increase in creatine kinase (CK) (12,482 U/L, range 53–138), and a marked increase in the catalytic activity of aspartate aminotransferase (AST) (238 U/L, 3–35) (Chiodini and Sundberg, 1982; Rosskopf et al., 1982; Frye, 1991b; Carpenter et al., 2001; Diethelm and Stein, 2006). Hyperphosphatemia and a decreased calcium/phosphorus ratio were suggestive of renal failure. This interpretation was corroborated by the increase in uric acid. The increased AST coupled with the increased CK was likely secondary to hepatic or muscular cytolysis, although it could also have been caused by unintentional aspiration of muscle tissue during cardiocentesis. Hyperproteinaemia with normoalbuminaemia suggested a hyperglobulinaemia consistent with hematopoietic neoplasia. Serum protein electrophoresis would have been indicated in this case, but no reference intervals for boa constrictors are published.

Oral and cloacal swabs sent to test (polymerase chain reaction [PCR]) for viruses known to cause neurological signs in snakes (Paramyxovirus, Reovirus, Adenovirus, Iridovirus) were negative (Wellehan and Johnson, 2005; Bennett and Mehler, 2006; Ritchie, 2006; Jacobson, 2007; Pees et al., 2010; Marschang, 2011).

To investigate the coelomic mass further, conscious radiographs were taken in left lateral and ventro-dorsal views. These revealed a diffuse opacity, about 3–4 cm long, situated a few centimeters cranial from the cardiac silhouette and causing a ventral deviation of the trachea (Fig. 2). The mass was distinctly separate from the heart, as can be seen on both views. Ultrasound, performed with a high-resolution 15-MHz linear probe (MyLab, Esaote, Via A. Siffredi, Genova, Italy) in a ventral approach (Silverman, 2006) with the animal manually restrained in dorsal recumbency, revealed a 1-cm-deep homogeneous mass; it was hypoechoic when compared to the underlying tissue. The echogenicity was not considered consistent with an abscess or granuloma, but more compatible with a solid neoplasm. The myocardium was also examined and did not present any abnormalities. No other abnormalities were detected, apart from the presence of moderate accumulations of hyperechoic crystals in both kidneys, suggestive of gout.

Because the mass did not have the echographic aspect of a vascular tumor, it was deemed safe to proceed to an ultrasound-guided fine-needle aspiration (FNA). The patient was so lethargic that it was considered safer to do the pro cedure without general anesthesia. An 8.8-MHz biconvex probe (MyLab) was used during the procedure. The mass cytology (7 slides, MGG stained) revealed a high density of cells mixed with red blood cells; these cells were mostly monomorphic large round cells characterized by round nuclei and moderate basophilic cytoplasm. The nucleus was sometimes irregularly shaped or lobed, often with 1 or more nucleoli. The cytoplasm sometimes had a clear perinuclear halo (archoplasm). These cells were immature lymphocytes and were mixed with a few heterophils and lymphocytes (Fig. 1B). Cytology showed a majority of atypical monomorphic round blast-like cells, consistent with a lymphoid hematologic malignancy: lymphoblastic lymphoma.

Finally, using the combination of all of these complementary diagnostic tests (hematology, diagnostic imaging, PCR, and cytology), the authors were able to reach a final antemortem diagnosis of lymphoblastic lymphoma, lymphoid leukemia, and IBD. These are summarized in Table 2. Given the severity of the clinical signs and the concomitant presence of 3 fatal diseases, the owner declined chemotherapy and elected euthanasia.

A postmortem examination was performed. Sections of the mass, liver, spleen, small intestine, colon, mandible, brain, lung, trachea, kidneys, and testis were collected in 10% neutral buffered formalin, processed routinely, embedded in paraffin, sectioned at 3–5 microns, mounted on glass slides, and stained with Harris hematoxylin and eosin. The firm mass cranial to the heart was adjacent to the esophagus and the trachea, at the level of the thymus. It was grossly whitish to diffuse pink, with small areas of hemorrhage that could have been caused by the FNA performed a few days previously. Histologically, it was composed entirely of neoplastic lymphoblasts and had considerable necrosis within deeper portions of the neoplasm. It contained a dense proliferation of medium-sized to large-sized round cells with a scant cytoplasm and a round nucleus containing a prominent nucleolus. Anisocytosis and anisocaryosis were moderate. Mitotic index was difficult to evaluate because of the necrosis but averaged 3 mitoses per high power field in preserved areas. These neoplastic cells infiltrated the esophageal muscle layer and the tracheal lamina propria. Large areas of necrosis and hemorrhage were observed within the mass. On the basis of anatomic topography and histology, it was suspected to be a thymic mass. An attempt at neoplastic cells identification was done by performing immunohistochemistry with anti-CD79a and anti-CD3 used for mammalians. Spleen from another boa was stained concurrently as a positive control. However, the protocol failed to positively label any cells in either the neoplastic mass or the normal spleen.

In the liver, large aggregates of neoplastic cells were observed in the portal areas, severely compressing adjacent hepatocytes. Splenic architecture was diffusely obliterated by sheets of neoplastic cells. The submucosa and the muscular layer of the intestine were multifocally and severely infiltrated by neoplastic cells. Subcutaneous adipose tissue contained a focally extensive accumulation of neoplastic cells similar to those previously described. In the brain, a large number of neurons and glial cells contained 1–3 round eosinophilic hyaline inclusions, 1–5 μm in diameter, in their cytoplasm that were associated with a mild neuronal necrosis and a diffuse moderate gliosis (Fig. 3). A few vascular lumina were filled with neoplastic cells similar to those previously described. Respiratory epithelial cells in the lungs presented numerous inclusion bodies in the cytoplasm. In the kidneys, glomerular tufts were diffusely distended by an accumulation of mesangial matrix associated with mild mesangial cell proliferation. The interstitium was multifocally fibrotic and mildly infiltrated with lymphocytes, plasma cells, and macrophages. Collecting ducts were multifocally dilated and contained in their lumen urate tophi admixed with desquamated epithelial cells. In the pericardium, adipose tissue was severely atrophic. In the testes, seminiferous tubules were diffusely atrophic; this was associated with moderate interstitial fibrosis. Epididymal cells contained inclusion bodies similar to those previously described. Histopathological examination showed multicentric atypical lymphoid proliferation (thymus, liver, spleen, brain, intestine) with lymphoid leukemia. Neuronal, glial, and epithelial intracytoplasmic inclusion bodies were conclusive of IBD. Moderate membranoproliferative glomerulonephritis with mild chronic interstitial nephritis and terminal renal gout were seen. Severe serous atrophy of pericardial adipose tissue and severe testicular atrophy were also noted.

Many reviews and retrospective case series on reptilian tumors have been published in the past 40 yr (Hashbarger, 1974; Elkan and Cooper, 1976; Hashbarger, 1976; Effron et al., 1977; Hashbarger, 1977; Hashbarger, 1979; Machotka, 1984; Frye, 1991a; Ramsay and Fowler, 1992; Frye, 1994; Cooper, 2000; Garner et al., 2004; Hernandez-Divers and Garner, 2004, Mauldin and Done, 2006). These include tumors of both the lymphatic and hematopoietic systems, such as lymphomas, leukemias, or both (Table 1).

Lymphoma can be diagnosed by cytological examination of FNA from a lesion. Some cases require biopsies, taken either during surgery or at necropsy. Lymphoma may sometimes induce leukocytosis (lymphocytosis, heterophilia), but, because large increases in white blood cells can be caused by septicemia, severe bacterial infections, or other chronic antigenic stimulation, these variations should not be used to provide a definitive diagnosis (Gyimesi et al., 2005; Folland et al., 2011).

A dozen cases of leukemia have been reported in reptiles (Table 1). The diagnosis of leukemia is based on CBC, blood smear cytology, and, if possible, a bone marrow biopsy. CBC can reveal nonregenerative anemia, thrombocytopenia, heteropenia caused by myelosuppression (cytopenias being more severe in acute rather than chronic leukemias), as well as the presence of atypical cells such as lymphoblasts. A leukemic blood profile should never be used to provide a definitive diagnosis; this can only be made from bone marrow biopsies or FNAs (Chiodini and Sundberg, 1982; Frye, 1991a; Morris and Dobson, 2001; Diethelm and Stein, 2006), which is impossible in ophidians (although it can be done in other reptile classes) without surgical removal of a rib under general anesthesia (Hernandez-Divers, 2006). Because of this limitation, leukemia may be suspected in snakes in cases of marked leukocytosis and when a significant percentage of atypical blood cells are seen on the cytological evaluation of a blood smear (Frye and Carney, 1973; Frye and Kass, 1990; Schultze et al., 1999; Tocidlowski et al., 2001; Raiti et al., 2002; Mauldin and Done, 2006).

Some authors have reported concomitant lymphoma and leukemia (Table 1). In a paper describing 8 Egyptian spiny-tailed lizards (Uromastyx aegyptia) diagnosed with lymphoma, 6 of the animals also had a CBC profile consistent with leukemia (Gyimesi et al., 2005). The relationship between leukemia or lymphoma and presence of a virus has been suspected in a few case reports (Jacobson et al., 1980; Chandra et al., 2001; Gyimesi et al., 2005; Schilliger et al., 2011).

Various treatments can theoretically be used alone or in association to treat malignant neoplasia in reptiles; nevertheless the prognosis for lymphoma and leukemia in reptiles is always poor. Feasible treatments include surgery (conventional, laser, or cryosurgery), radiotherapy, photodynamic therapy, and chemotherapy (Frye, 1994; Rosenthal, 1995; Garner et al., 2004; Hernandez-Divers and Garner, 2004; Reavill, 2004; Mauldin and Done, 2006; Georoff et al., 2009; Folland et al., 2011). Surgery is only indicated for solid tumors with low metastatic potential. Only a few publications discuss treatment regimens in reptiles, and little information is available on the use of chemotherapy to treat lymphoma and/or leukemia in these animals (Jacobson et al., 1981; Silverstone et al., 2007; Georoff et al., 2009; Folland et al., 2011; Jankowski et al., 2011). Difficult venous access, lack of specific treatment protocols (dosage, time intervals), and difficulty in monitoring systemic effects of treatment are only some of the limitations of chemotherapy in snakes. The effect of cytotoxic drugs on white blood cell counts (and hence immunity) in reptilian patients is also unknown. Treatments have been sporadically attempted, but most patients had to be euthanized on welfare grounds. A therapy of cytosine arabinoside was tried unsuccessfully on a rhinoceros viper (Bitis nasicornis) suffering from lymphoma (Jacobson et al., 1981). A diamondback terrapin (Malaclemys terrapin) with acute lymphoid leukemia was treated with a combination of chlorambucil, cytosine, and prednisolone, but despite an improvement 24 h posttreatment, the animal died after 46 days (Silverstone et al., 2007). Jankowski and colleagues (2011) treated a 4.5-year-old male bearded dragon (Pogona vitticeps) diagnosed with monocytic leukemia with cytosine. The animal died 48 h after the start of treatment. A 13-yrold green tree monitor (Varanus prasinus) with chronic T-cell lymphoid leukemia was treated with prednisolone for 22 days, followed by a single dose of prednisolone and chlorambucil 184 days later (Georoff et al., 2009). The animal died a few days after this second treatment. Interestingly, in a 2-year-old female green iguana (Iguana iguana) diagnosed with lymphoma in the neck region, a single radiotherapy session (10 Grays) associated with doxorubicin, vincristine, cyclophosphamide, and prednisone treatment successfully managed lymphoma for at least 2.5 yr (Folland et al., 2011). Intralesional injections can offer an easier alternative but are only possible with solid tumors.

Although IBD is thought to be of a viral etiology, the causative agent has not yet been conclusively identified. Based on histological observations, various studies have postulated that a retrovirus may be the possible etiological agent, but no disease transmission studies have been performed to confirm this hypothesis. To date, Koch's postulates have not been fulfilled to conclude a relationship between retrovirus and IBD (Wozniak et al., 2000; Jacobson et al., 2001; Wellehan and Johnson, 2005; Ritchie, 2006; Pees et al., 2010; Marschang, 2011). Furthermore, healthy boa constrictors have been shown to harbor endogenous retroviruses (Martin et al., 1997) and a novel endogenous retrovirus has been partially sequenced from healthy blood pythons (Python curtus), clinically healthy Burmese pythons (Python molurus bivittatus), and Burmese pythons with clinical signs of IBD (Huder et al., 2002). Inclusion bodies have also been found in various tissues of clinically healthy animals (Orós et al., 1998; Jacobson et al., 2001; Raymond et al., 2001; Schumacher, 2006; Vancraeynest, 2006; Jacobson, 2007; Chang and Jacobson, 2010; Keilwerth et al., 2012).

In a recent paper, Steinglein and colleagues (2012) used a metagenomic approach to search for a possible etiologic agent in snakes with confirmed IBD. The study isolated the complete genomic sequences of 2 viruses related to arenaviruses and a third arenavirus-like sequence. Of the 8 snakes confirmed with IBD, RNA virus was detected in 6 of them and none was found in 18 healthy snakes. The isolated viruses have a typical arenavirus genome organization but belong to a separate lineage from Old and New World arenaviruses. In addition, the genomes also encode envelope glycoproteins that are more similar to filoviruses than arenaviruses. Currently, no vaccines are available for IBD and euthanasia is indicated for confirmed cases because IBD is not curable, and supportive treatment does not alter the course of the disease. If Koch's postulates could be fulfilled, demonstrating the arenavirus as the causative agent of IBD, arenavirus-targeting vaccines or treatments could be effective against snake IBD (Stenglein et al., 2012).

Clinical signs of IBD are variable and nonspecific. They include lethargy, anorexia, regurgitation, weight loss, stomatitis, secondary infections, and neurological symptoms (incoordination, disorientation, head tremors, loss of righting reflex). Pneumonia, dermatitis, cutaneous sarcomas, leukemia and lymphoproliferative disorders have also been reported in some cases (Carlisle-Nowak et al., 1998; Chang and Jacobson, 2010; Orós et al., 1998; Schilliger et al., 2011; Schumacher, 2006; Schumacher et al., 1994; Vancraeynest et al., 2006). The exact route of transmission is unknown, although horizontal (body fluids, venereal, and fecal/oral routes) and vertical (placental or through eggs) transmission is suspected. The snake mite (Ophionyssis natricis) may also act as vector in horizontal transmission (Schumacher et al., 1994; Carlisle-Nowak et al., 1998; Orós et al., 1998; Wellehan and Johnson, 2005; Schumacher, 2006; Vancraeynest et al., 2006; Chang and Jacobson, 2010; Pees et al., 2010; Marschang, 2011; Schilliger et al., 2011).

The detection of viruses in reptiles relies on a wide range of tools, including cell culture, molecular methods (PCR, reverse-transcriptase [RT]-PCR, sequencing) as well as metagenomics. Antemortem confirmation of IBD is challenging, as there is no specific clinically applicable diagnostic method. Diagnosis is obtained by demonstrating the characteristic inclusion bodies in biopsies from internal tissue, most commonly from liver or esophageal tonsils (Schumacher et al., 1994; Carlisle-Nowak et al., 1998; Orós et al., 1998; Vancraeynest et al., 2006; Chang and Jacobson, 2010). Inclusion bodies can also be observed in the cytoplasm of circulating erythrocytes, lymphocytes, and heterophils (stained with hematoxylin and eosin [H&E] or Diff-Quick), but this diagnostic method has a poor sensitivity compared to histopathology of hepatic biopsies (Schumacher, 2006; Jacobson, 2007; Banajee et al., 2012; Keilwerth et al., 2012).

Postmortem diagnosis is based on the presence of characteristic eosinophilic or amphophilic intracytoplasmic inclusions in various organs. In boa constrictors, inclusions are commonly found in epithelial cells lining the esophageal tonsils, gastrointestinal and respiratory tracts, as well as in hepatocytes and tubular epithelial cells (Schumacher et al., 1994; Jacobson, 2007). Inclusion bodies can also be found in neurons. Wozniak and colleagues (2000) showed that the inclusions were nonviral and contained a unique 68kD protein. It is not clear whether the inclusions are directly caused by infection. Euthanasia is indicated for confirmed cases because IBD is not curable and supportive treatment does not alter the course of the disease. No vaccines are available and the only prevention involves snake mite control and rigorously quarantining of new arrivals.

This case presents an uncommon combination of 3 processes in a single animal: a hematopoietic neoplasia, a lymphoma, and IBD. It closely resembles another case concerning a red tail boa (Boa constrictor constrictor) that was presented with a leukemic blood profile, lymphoblastic lymphoma, and IBD (Schilliger et al., 2011). The differences between these 2 cases resided in that 1) the diagnosis of the present case was determined antemortem, whereas the former case was diagnosed postmortem (the snake died immediately following blood sampling via cardiocentesis and no imaging technique could be performed), and 2) the former case was presented with an even higher leukocytosis (820 × 10³/μL vs. 102 × 10³/μL). In all other aspects, both cases were strikingly similar, and as in the present case the thymus of the Boa constrictor constrictor was completely effaced by neoplastic lymphoblasts.

Lymphoid leukemia differs from malignant lymphoma primarily in the anatomic distribution. Solid neoplastic masses are present in lymphoma but are less common in patients with primary lymphoid leukemia (Thrall et al., 2004). Nevertheless, leukemic blood and bone marrow are frequently found in dogs with lymphocentric lymphoma (Raskin and Krehbiel, 1989; Thrall et al., 2004). It is unfortunate that we were unable to proceed to a bone-marrow biopsy in order to confirm the diagnosis of lymphoid leukemia. This constitutes the main limitation of this case. In snakes, bone-marrow biopsies can only be taken by removing a rib under general anesthesia, but this would have constituted a significant risk for this patient (Hernandez-Divers, 2006). However, the marked leukocytosis (WBC: 102.0 × 10³/μL), lymphocytosis (91.8 × 10³/μL), and presence of atypical cells was highly suggestive of leukemia. Furthermore, the 3 independent laboratories that performed the diagnostic tests (blood smear cytology, IBD testing, postmortem histopathology) confirmed a lymphoid leukemia without the need for separate bone-marrow biopsies. It remains unclear whether the neoplasms began their proliferation within the bone marrow or whether leukemia was a feature of disseminated, end-stage lymphoma.

The second limitation of this case was the failure to identify the neoplastic cell lineage (T or B cell) of the lymphoma. Immunohistochemistry performed with the use of anti-CD79a and anti-CD3, which are commonly used for domestic animals, failed to identify neoplastic cells. Immunophenotyping of lymphocytes is being increasingly described in reptiles, but most of the articles found in the literature use cytochemical staining (Tocidlowski et al., 2001; Silverstone et al., 2007; Georoff et al., 2009), flow cytometry (Conrad et al., 2007), or transmission electron microscopy (Orós et al., 2001; Gyimesi et al., 2005); none of these were available for this case. Immunohistochemistry for B and T lymphocytes with anti-Pax 5 and anti-CD3 antibodies has been reported in a Chinese Box turtle (Cuora flavomarginata) (Bezjian et al., 2013) and a Diamond python (Morelia spilota spilota) (Raiti et al., 2002). Again, both of the protocols described in these articles were not available for this case.

Furthermore, it was not possible to qualify the exact origin of the cervical mass from the FNA and postmortem biopsies. The pathologist was unable to identify any thymic cells, possibly because they had all been replaced by neoplastic cells. However, the location of the mass was highly suggestive of a thymic infiltration. The authors decided not to perform a liver biopsy because of the positive result for IBD on blood cytology. Indeed, the diagnosis of IBD based on blood smears has a high specificity (low number of false positives) and low sensitivity (the absence of inclusion bodies does not allow the exclusion of IBD, high number of false negatives) (Carlisle-Nowak, 1998; Orós et al., 1998; Wozniak et al., 2000; Jacobson et al., 2001; Raymond et al., 2001; Schumacher, 2006; Vancraeynest et al., 2006; Chang and Jacobson, 2010; Keilwerth et al., 2012). Hence, the positive result allowed the authors to avoid a biopsy (to be performed under general anesthesia), which would have been a significant risk in this patient. Considering that IBD had been confirmed and that the other viral PCR tests came back negative, the authors did not feel it was justified from a financial point of view to ask the owner to pursue further investigations, such as electron microscopy. Moreover, it would have been beneficial to perform protein electrophoresis in order to differentiate between a polyclonal (inflammation, infection) and monoclonal (hematopoietic neoplasia) hyperglobulinaemia. Finally, it would have been interesting to attempt chemotherapy on this patient, even if the current published protocols are random and disparate.