Monocytic Leukemia with Tissue Metastases in a Bearded Dragon (Pogona vitticeps)

Deidre K. Fontenot,¹ Christopher R. Gregory,² Nadine Lamberski,¹ Raymond P. Campagnoli,³ and Kenneth S. Latimer³

¹ Riverbanks Zoological Park and Botanical Gardens, P. O. Box 1060, Columbia, SC 29202-1060; ² Department of Small Animal Medicine and ³Department of Pathology, College of Veterinary Medicine, The University of Georgia, Athens, GA, USA

Abstract: A five-year-old, captive-bred, male bearded dragon (Pogona vitticeps) was presented for lethargy, weight loss, and dehydration. Initial hematology and biochemistry tests revealed a severe leukocytosis (77,000 leukocytes /µl), an increased aspartate aminotransferase (AST) activity (453 IU/L), and a hypercholesterolemia (500 mg/dl) as compared to the International Species Information System (ISIS) reference ranges. ¹ Blood cultures were positive for Clostridium sp. Monocytoid infiltrates were observed in an initial liver biopsy. Follow-up hematology showed a continued leukocytosis with a majority population of mature and immature monocytes. Based on these findings, a diagnosis of metastatic monocytic leukemia was made. After ten months of supportive care, the animal was euthanized due to declining quality of life. Post-mortem histopathology showed metastasis of monocytes to the periocular region, liver, kidney, and intestines. This is the first report of monocytic leukemia with tissue metastases in a reptile.

Key Words: Reptile, Bearded dragon, Pogona vitticeps, Monocytic leukemia, Leukemia, Sepsis, Tissue metastasis, Myelogenous sarcoma, Tumor

Case Report

A five-year-old, captive-bred male, bearded dragon (Pogona vitticeps) was evaluated for lethargy, weight loss, and dehydration. The animal was housed in a naturally lighted multiple species exhibit with other desert lizards. The group was offered a daily mixed salad with multivitamin supplementation. Bearded dragons in the exhibit were offered pinkie mice once weekly and multivitamin-covered crickets twice weekly. This animal had a medical history of intestinal parasitism and loss of one of digit during territorial aggression within the exhibit.

Subjective findings included 7- 10% dehydration, thin body condition, caseous discharge from the left commissure of the mouth, and firm digital swelling of the left front foot. The remainder of the physical exam was unremarkable. Objective findings included a severe leukocytosis of 77,000 cells/µl (reference interval = 9,381 ± 5,024 cells/µl). Blood cultures were positive for Clostridium sp. Serum biochemistries showed an increased aspartate aminotransferase (AST) activity (453 IU/L; reference interval = 27 ± 19 IU/L) and hypercholesterolemia (500 mg/dl; reference interval = 211 ± 210 mg/dl). ¹ Changes consistent with bone lysis were observed in radiographs of the swollen digit. Supportive care with fluid therapy and intramuscular (IM) antibiotics (ceftazidime^a at 25mg/kg every 48 hours) was implemented until the animal was stable for surgical amputation of the infected digit and debridement of the commissure abscess. Based on the biochemical findings, a keyhole liver biopsy was also performed during surgery. The procedures were performed under propofol^b induction (10mg/kg intravenously) and isoflurane^c anesthesia via endotracheal intubation.

Clear yellow coelomic fluid was obtained during the liver biopsy procedure. The fluid was a borderline transudate (protein = 2.6 g/dl, no cells on sediment examination) and was later negative on cultures for aerobic and anaerobic organisms. Fresh biopsy samples were used for microbial cultures and the remaining liver was placed in 10% neutral-buffered formalin and submitted to a pathologist (Gregory) for histopathologic evaluation. Aerobic and anaerobic cultures of the transudate and liver tissue were

negative for microorganisms. Microscopic examination of the liver biopsy revealed severe multifocal to coalescing infiltrates of cells in periportal areas. The cells were monocytoid with oval to cleaved to reniform nuclei and moderate, occasionally vacuolated, cytoplasm. Organisms were not observed with routine special stains (Wright's-Giemsa, periodic acid-Schiff, Kinyoun's acid-fast) of replicate liver sections. Probable tissue metastasis secondary to monocytic leukemia was diagnosed. Wright's-stained and unstained peripheral blood smears were requested by the pathologist for evaluation.

The predominant leukocytes on the stained blood smears were mature and immature monocytes (Fig. 1). A smaller population of leukocytes contained low numbers of intracytoplasmic granules typical of immature heterophils. The immature monocytes contained round to oval to indented nuclei with a fine chromatin pattern and occasional nucleoli. The nuclear to cytoplasmic ratio was increased. The cytoplasm was generally basophilic and occasionally vacuolated. Unstained smears were submitted for cytochemical analysis using commercially available kits for human blood cells. ^d Monocytoid cells were negative for lipid with Sudan black B stains. These cells also were negative for peroxidase, acid and alkaline phosphatase, and naphthol AS-D chloroacetate activities. However, the cells were positive for alpha naphthyl acetate (non-specific) esterase activity (Figs. 2a,b). Non-specific esterase activity was inhibited with sodium fluoride treatment in the majority of cells (Fig. 3). Based on reported staining characteristics of other reptilian blood cells^2,3 and comparative results in mammalian cells⁴, a presumptive diagnosis of monocytic leukemia was made.

Fig. 1. The majority of leukocytes in the peripheral blood smear were mature and immature monocytes. These cells contained basophilic to blue-gray cytoplasm. Nuclear morphology ranged from round to cleaved to oblong with a fine chromatin pattern. Wright’s stain, magnification = 1,000x.

Fig. 2a. Low power magnification of a peripheral blood smear showing the positive reaction (golden brown) with alpha naphthyl acetate (non-specific) esterase. The monocytes were strongly reactive. Nonspecific esterase technique with fast green counterstain, magnification = 100x.	Fig. 2b. Higher magnification of figure 2a. Magnification = 500x.

Fig. 3. Inhibition of non-specific esterase activity with sodium fluoride. The remaining positive cells were identified as lymphocytes. Magnification = 200x.

Supportive care included hyperalimentation with salad and insects and daily soaking with subcutaneous (SQ) fluid therapy (40 ml/kg lactated ringers) every 72 hours. Systemic antibiotic therapy was continued with intramuscular ceftazidime^a (25 mg/kg every 72 hours) for 15 days followed by intramuscular ampicillin^e (5mg/kg once daily) for 14 days in combination with intramuscular amikacin^f (2.5 mg/kg every 72 hours). The animal continued to improve in attitude, activity level, and body weight for six months after the presumptive diagnosis of leukemia was made. However, the CBC showed minimal improvement with a continued increase in peripheral monocyte count (43,000 cells/µl), an overall leukocytosis (91,600 cells/µl), and a nonregenerative anemia (hematocrit =12%). Evaluation of the serum chemistry panel revealed a marked decrease in the AST activity (47 IU/L) to within ISIS reference ranges. However two months later (8 months from diagnosis of leukemia), the keeper reported an acute swelling of the left periocular region. An external ophthalmic exam was difficult due to the severity of the periocular swelling. Ultrasound examination of the globe revealed a possible hyperechoic intraocular mass within the vitreous humor and enucleation of the left eye was elected under isoflurane^c anesthesia. A periocular caseous exudate was noted during the enucleation procedure. Culture of the exudate later revealed Proteus mirabilis and Pseudomonas aeruginosa. An intraocular culture was not performed. Histopathology of the eye showed severe multifocal hemorrhage of the inner conjunctival sac with multifocal aggregates of monocytoid cells admixed with heterophils. The cells were present within an eosinophilic, slightly fibrillar, matrix. An occasional heterophilic granuloma also was present within the matrix. Both the granulomas and the matrix contained bacilli. The edematous connective tissue stroma also contained mild multifocal infiltrates of the monocytoid cells, indicating possible tissue metastasis of the leukemia. Despite continued supportive treatment and antibiotic therapy, the reptile's quality of life declined further and euthanasia was elected 10 months after the initial diagnosis of monocytic leukemia.

On gross necropsy examination, proteinaceous fluid was noted periocularly at the enucleation site as well as in the subcutaneous tissue of the neck, ventral coelomic wall, and coelomic cavity. Fluid was submitted for aerobic and anaerobic culture. The liver was small and dark purple with coalescing white foci. The bone marrow appeared fatty in appearance. Representative tissue sections were placed in 10% neutral-buffered formalin and submitted to the pathologist for histopathologic evaluation. Wright's-stained bone marrow smears were evaluated in-house. Microscopically, the bone marrow was acellular and interpreted to be terminal myelofibrosis secondary to the leukemia. Histopathology of the liver revealed effacing of the normal hepatic architecture with anastomosing chords and clusters of monocytoid cells (Fig. 4). Similar infiltrates were observed within the kidneys, the intestinal submucosa and muscularis, and the edematous subconjunctival tissue. All cultures were negative for microorganisms. A final diagnosis of monocytic leukemia with tissue metastasis was made.

Fig. 4. Sections of liver contain marked multifocal infiltrates of monocytoid cells that replaced and disrupted the normal hepatic architecture. Similar cells were observed in other tissues. H&E stain, magnification = 1000x.

Discussion

The neoplasm in this case report appears to be the first reported case of metastatic monocytic leukemia in a reptile. Several cases of hematopoietic neoplasia in reptiles have been reported in the literature. Lymphomas, leukemias, and other myeloproliferative disorders were reported in lizards,^{5-11, 20, 21} squamates,^{5,9,10,12-18,20,21} and Chelonians. ^{4,5,19,20, 21} One report of lymphoblastic leukemia in a bearded dragon also was found. ⁷ Malignant lymphoma (lymphosarcoma) represented the majority of the documented cases of myelogenous neoplasms in reptiles. Typical metastases to other tissues such as the liver, myocardium, and kidneys were observed. Many of the cases initially presented with cutaneous tissue masses that were found to be metastatic myeloproliferative disease. None of the cases reviewed were noted to have secondary sepsis; however, many of the cases were diagnosed post-mortem. Furthermore, no reports of life expectancy after diagnosis were noted in the literature. This is also likely due to the large number of post-mortem diagnoses.

This case report illustrates a thorough diagnostic evaluation for lethargy and dehydration in a bearded dragon with a clinical diagnosis of monocytic leukemia with tissue metastases. Histopathology and cytochemistry were essential in this diagnosis. Hematopoietic neoplasia is a prominent disease process in reptiles that must always be taken into consideration.

The clinical signs in the bearded dragon were nonspecific. The elevated AST activity and hypercholesterolemia indicated hepatocyte damage and dysfunction. Both abnormalities warranted the liver biopsy. Histopathologic evaluation of the liver and follow-up evaluation of peripheral blood smears were keys in the diagnosis of the primary disease. The biopsy also complemented the peripheral blood evaluation and was a key prognostic indicator in this case. Because of the evidence of metastatic disease at the time of diagnosis of monocytic leukemia, the animal’s prognosis was poor. The progression of disease seemed to correspond with the increase in the peripheral monocyte count as well as the development of the nonregenerative anemia. The anemia was likely due to myelofibrosis. Myelofibrosis is a common "end stage" of chronic myeloproliferative diseases in domestic animals. ⁶

Cytochemical evaluation of the cells in the peripheral blood smear was essential in characterizing the type of leukemia. Although staining characteristics of reptilian blood cells may vary, the staining characteristics of the cells in the bearded dragon closely resembled those of monocytes in some reptiles and in mammals. Blood samples were not available for evaluation with transmission electron microscopy. Ultrastructural characteristics of cytoplasmic structures may have aided in differentiation of leukemias of monocytic, myelocytic, or myelomonocytic origin.

In domestic animals, monocytic leukemia is extremely rare. ^6,22 This myeloproliferative disorder tends to invade the bone marrow, spleen, lymph nodes and sometimes liver with rare metastasis to kidneys, heart and lungs. ²² Affected individuals usually have a non-regenerative anemia and fever due to a marked leukocytosis. Death is usually due to secondary infection or hemorrhage. Most cases have a poor prognosis with a quick entry into a terminal blast crisis. ²²

Few cases of treatment regimes for neoplasms have been discussed in the literature for exotic animals,^23,24 especially reptiles. ^11,15 Surgical excision of cutaneous masses and supportive care appear to be the most common treatment modality discussed in the literature. ⁵ Some treatment modalities routinely used in human and domestic animals were discussed in the literature for use in reptiles. ^5,18 These treatment options included radiation therapy, chemotherapy, and photodynamic therapy. Lack of readily accessible peripheral vessels for intravenous administration of chemotherapeutic agents, lack of specific treatment protocols (dosages, time intervals, etc. ), and difficulty in monitoring the systemic effects of the treatment are some of the limitations in use of these modalities in reptiles. ⁵ Therapeutic options considered in this case included supportive care versus one or more chemotherapeutic regimens of cyclophosphamide and/ or aspariginase; vincristine and prednisone; oral hydroxyurea; or oral busulfan. ^5,6,25,26 With evidence of metastatic disease at the time of diagnosis, supportive care was elected.

Sources and Manufacturers

^aFortaz (ceftazidime sodium), Glaxo-Wellcome, Inc. , North Carolina, USA
^bPropoflo (propofol), Abbott Laboratories, North Chicago, Illinois, USA
^cIsoflo (isoflurane), Abbott Laboratories, North Chicago, Illinois, USA
^dSigma Diagnostics, St. Louis, MO, USA
^eOmni-pen (ampicillin sodium), Wyeth-Ayerst Laboratories, Philadelphia, Pennsylvania, USA
^fAmiglyde-V (amikacin sulfate), Fort Dodge Laboratories, Inc. , Fort Dodge, Iowa, USA

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