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Lymphosarcoma with Lymphoid Leukemia in an Aruba Island Rattlesnake, unicolor

Brad Lock1, DVM, Darryl Heard1, BVMS, PhD, DACZM, Daniel Dunmore2, DVM, MS, Shashi Ramaiah3, DVM, MS, PhD

l! Department of Small Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610, USA 2. Department of Pathobiology, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610, USA Downloaded from http://meridian.allenpress.com/jhms/article-pdf/11/4/19/2206983/1529-9651_11_4_19.pdf by guest on 29 September 2021 3. Department of Physiological Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610, USA

Abstract: Abstract: A six year old male, Aruba Island rattlesnake, Crotalus unicolor, with diffuse cranial swelling and anorexia was diagnosed with multicentric lymphosarcoma with lymphoid leukemia. Significant hematologic findings included a marked leucocytosis (137,000), monocytosis and basophilia with marked het- eropenia and mature lymphopenia. The predominant (95.5%) white cells were large blast-like cells. Cytological examination of a tissue aspirate from the head revealed a highly cellular monomorphic population of round mononuclear cells. Histologic examination revealed that most parenchymal organs and tissues con­ tained monotonous sheets of neoplastic lymphoid cells.

Key Words: Aruba Island rattlesnake, Crotalus unicolor, hematopoietic, lymphoma, lymphoid, leukemia, neo­ plasm.

INTRODUCTION then grasped behind the head and a syringe case was placed over the head and between the maxilla and mandible to cover Although an important cause of morbidity and mortality in the fangs and to protect against accidental envenomation. The domestic (Moulton, 1990), neoplasia has been histor­ animal was then intubated and anesthesia was maintained ically less commonly documented in (Jacobson, et al, with 3% isoflurane in oxygen. 1980). Prior to 1962 there were no reptilian hematopoietic Physical examination showed marked cranial swelling and system neoplasias reported in the literature (Lawson, 1962, edema, and pale, edematous mucous membranes (Figure 1A Dawe, 1969) and there has been only one report of a lym­ and IB). A 1.0 cm diameter soft tissue mass was palpated phosarcoma and/or leukemia in a rattlesnake approximately two thirds of the body length from the head. A (Harshbarger, 1974). Recent reports (Catao-Dias, 1999, heparinized blood sample was collected from the ventral coc­ Ramsey, et al, 1996) have found neoplasms in 12 - 23% of cygeal vein and submitted for a complete blood count (Table necropsies performed on snakes. This is the first report of a 1) and plasma biochemical panel. The body was also radi­ neoplastic disease in the endangered Aruba Island rattlesnake, ographed and a fine needle aspirate from the swollen head Crotalus unicolor. was submitted for cytological examination. Significant hematologic findings included a marked leuco­ CASE REPORT cytosis (137,000/pl) characterized by large numbers of blast-like cells, monocytosis and basophilia with marked het- A six-year-old, 1.0 kg, adult, male Aruba Island rat­ eropenia and mature lymphopenia. The predominant (95.5%, tlesnake, Crotalus unicolor, was presented to the Veterinary 130,800/pl) white cells were large blast-like cells with moder­ Medical Teaching Hospital (VMTH), University of Florida, ate light blue cytoplasm, fine nuclear chromatin and indistinct because of diffuse cranial swelling and anorexia. The swelling nucleoli (Figure 2). Occasional mitotic figures were seen in was first noticed l i d prior to presentation and had remained the blast-like component. Although thrombocyte numbers unchanged. The animal was housed with another male Aruba appeared decreased, this was attributed to significant clump­ Island rattlesnake in a separate enclosure in a large mixed col­ ing rather than true thrombocytopenia. The plasma lection of at the Central Florida Zoo. The was biochemical profile showed hypoglycemia (18 mg/dl, refer­ fed one small rat every two weeks, but it had refused the meal ence range mean (RRM), 64 mg/dl) mild hyperuricemia (23.3 offered 14 d prior to presentation. Visual examination mg/dl, RRM, 3.6 mg/dl) and increased alkaline phosphatase revealed the animal was in good body condition but it’s strik­ activity (150 U/L, RRM, 76 U/L). ing and rattling responses were depressed during Radiographic evaluation revealed adequate bone density. A manipulation. lateral view showed a round soft tissue opacity in the mid­ For anesthetic induction, the snake was placed with a snake body region at the caudal edge of the air sac. A rounded hook into a plastic induction chamber and administered 5% margin was observed in the same area on the ventro-dorsal isoflurane in oxygen until non- responsive. The snake was view. An approximately 5.0 mm in diameter soft tissue

Volume 11, No. 4,2001 Journal of Herpetological Medicine and Surgery 19 Table 1. Complete blood count of an Aruba Island rattlesnake, Crotolus unicolor, with lymphosarcoma and lymphoid leukemia.

Parameter Result

Red blood cells (xl03/pL) 0.64

Hemoglobin g/dl 6.1

Hematocrit % 28.4

MCV fl 443.8

MCH pg 95.3

MCHC g/dl 21.5

White blood cells (x103/|jL) 137.0 Downloaded from http://meridian.allenpress.com/jhms/article-pdf/11/4/19/2206983/1529-9651_11_4_19.pdf by guest on 29 September 2021 Figure 1A. On physical exam the Aruba Island rattlesnake, Blast cells (xl03/pL) 130.8 Crotalus unicolor, had marked cranial swelling.

Heterophils (xl03/|jL) 0

Lymphocytes (xl03/pL) 0

Monocytes (xl03/pL) 4.1

Eosinophils (xl03/|jL) 0

Basophils (xl03/pL) 2.1

swelling was seen protruding from the right side of the head. There was an abrupt slope on the dorsal aspect of the head with a mottled, soft tissue opacity overlying this region. Cytological examination of the tissue aspirate from the head (Figure 3) revealed a highly cellular monomorphic pop­ ulation of round mononuclear cells which exhibited moderate variability in nuclear to cytoplasmic (N: C) ratio, anisocyto- sis, anisokaryosis, prominent nucleoli and lacy diffuse chromatin. A few cells had nuclei suggestive of plasmacytoid cells. The cytological interpretation was lymphosarcoma. Euthansia was performed for humane reasons using an over­ F igure IB. On physical exam the Aruba Island rattlesnake, dose of barbiturate (Buthanasia) and the snake was Crotalus unicolor, had edematous, pale mucus membranes. necropsied.

NECROPSY tinct borders and scant eosinophilic cytoplasm. The nuclei were round and located centrally with vesicular to coarsely Gross pathological examination revealed the snake to be in stippled chromatin and prominent nucleoli. Mitotic figures good physical condition with moderate subcutaneous fat averaged four per high powered field. Extensive areas of cel­ stores. There was a single well circumscribed, semi firm, 3.5 lular necrosis were also common. The coelomic mass was x 2 x 2.5 cm, uniformely tan mass present within the mid considered to be the spleen and was composed of dense coelomic cavity adjacent to the mildly distended gall bladder sheets of neoplastic cells supported by a fibrovascular stroma. (Figure 4). The liver was dark red to brown and contained two The skeletal muscle of the caudal aspect of the head was dif­ 0.3 cm diameter, firm, pale tan, nodular foci extending from fusely infiltrated by abundant neoplastic lymphocytes. These the capsule into the parenchyma. The kidneys were uniformly neoplastic cells were within a fibrous stroma admixed with a covered by numerous fine white streaks (urate stasis) that finely granular amphophilic material surrounding intact bun­ radiated from superficial veins and extended 0.3 cm into the dles of skeletal muscle. renal parenchyma. Select soft tissues were fixed by immersion in 10% neutral DISCUSSION buffered formalin for histologic examination and were sec­ tioned at 4 pm and stained with hematoxylin and eosin The initial differential diagnoses for the cranial swelling (H&E). Histologic examination revealed most parenchymal were stomatitis, venom gland infection, bite trauma or enven- organs and tissues including the liver, kidney, lung, gastroin­ omation from the cage mate, hypoproteinemia or congestive testinal tract, heart, adrenal glands, testicles, thyroid gland heart failure. Differential diagnoses for the coelomic mass and vessel lumina contained monotonous sheets of neoplastic were gall bladder, pancreas, spleen or neoplasia. The initial lymphoid cells (Figure 4B). These cells were round with dis-

20 Journal of Herpetological Medicine and Surgery Volume 11, No. 4,2001 Figure 3. Photomicrographs of a fine-needle tissue aspirate Downloaded from http://meridian.allenpress.com/jhms/article-pdf/11/4/19/2206983/1529-9651_11_4_19.pdf by guest on 29 September 2021 Figure 2. Photomicrograph of a peripheral blood smear (magnifi­ (magnification 250X) from a head swelling in the Aruba Island cation, 250X) from this Aruba Island rattlesnake, Crotalus rattlesnake, Crotalus unicolor, stained with Romanowsky-type unicolor, stained with Romanowsky-type stain (Wright-Geimsa). stain (Wright-Geimsa). Notice the monomorphic population of Notice the large blast cells with moderate light blue cytoplasm round mononuclear cells with lacey diffused nuclear chromatin with fine nuclear chromatin pattern and indistinct nucleoli. Two pattern, moderate N:C ratio with variable and indistinct nucleoli. thrombocytes with clear cytoplasm are present. The morophologic features are similar to blast cells noted in the peripheral blood smear. work up, consisting of a complete physical exam, complete blood count, plasma chemistry profile, radiographs and cyto- marrow or was a feature of terminal lymphosarcoma because logical examination of a tissue aspirate of the cranial swelling the bone marrow was not examined histologically. provided sufficient information for a rapid diagnosis and There are few reports of neoplasia in rattlesnakes and only prognosis in this case. one with a lymphoid and/or hematopoietic origin The Aruba Island rattlesnake in this report had advanced (Harshbarger, 1974). Neoplastic disease has been reported to neoplastic disease of hematopoietic origin characterized by be observed in 2 - 17.5% and 12.4 - 23.1 % of and leukemia and disseminated solid tissue involvement, either snake necropsies respectively (Effron, et al, 1977, Montali, via metastisis or multicentric development of the neoplasm. 1980, Ramsey, et al, 1996, Catao-Dias, 1999). Lymphoid The predominant cell type in the leukemia, (95.5%), was a and/or hematopoietic origin neoplasm’s accounted for lymphoblast-like cell. There was an associated heteropenia, between 0 and 30.8% of reptile cancers (Griner, 1983, basophilia and monocytosis. This leukogram resembles those Ramsey, et al, 1996, Catao-Dias, 1999). In a recent report, reported in a California kingsnake and a rhinoceros viper with involving 95 captive snakes over a ten year period, there were lymphosarcoma (Jacobson, et al, 1980, Jacobson, et al, no lymphoid neoplasms identified (Ramsey, et al, 1996). 1981). The heteropenia and mature lymphopenia is probably However, in another report neoplasms of lymphoid origin due to meylopthisis associated with the neoplasm (Schultze, were the most common (30.8%) neoplasm seen and multicen­ et al, 1999); however, bone marrow was not examined histo­ tric lymphosarcoma has been reported in a variety of snake logically in this case. The moderate hyperuricemia was most species from several families (Catao-Dias, 1999). The exten­ likely due to renal tubular dysfunction and necrosis secondary sive infiltration of parenchymal organs is consistent with that to neoplastic infiltration of the renal parenchyma. The moder­ reported in other reptiles. Reports of multicentric lymphosar­ ate hypoglycemia can be attributed to hepatic dysfunction coma and lymphoproliferative disorders include a young secondary to neoplastic infiltrate. Similarly, the increased savannah monitor, Varanus exanthematicus, a green iguana, alkaline phosphatase activity may have been due to cell leak­ Iguana iguana, a spiny tailed agamid, Uromastix acanthinu- age in organs damaged by the neoplasm. Subcutaneous soft rus, a Bengal monitor, Varanus bengalensis, a California tissue swelling in snakes with lymphoid neoplasms is uncom­ kingsnake, Lampropeltis getulus calforniae, e astern mon (Jacobson, et al, 1980, Jacobson, et al, 1981). However, kingsnake, Lampropeltis getulus getulus, and a rhinocerous soft tissue swelling of the neck and thoracic region was viper, Bids nasicornis, (Harshbarger, 1974, Jacobson, et al, described in a green iguana and a lacertid with lym­ 1981, Jacobson, 1981, Jacobson, et al, 1980, Montali, 1980, phosarcoma (Lawson, 1962, Romagnanao, et al, 1996). In the Goldberg, 1991, Catao-Dias, 1999, Schultze, et al, 1999). The Aruba Island rattlesnake, the head, fang sheaths and mucus cytological and histopathologic features of the neoplastic membranes were markedly swollen. This may be due to a lymphoid cells in this animal resemble those described in a combination of lymphatic drainage obstruction and/or neo­ California and eastern kingsnake, a rhinoceros viper, Indian plastic infiltrate. Classification of the leukemia as acute or python, Python molurus, and a boa constrictor, Boa constric­ chronic was not possible in this case. Serial blood profiles to r, with lymphosarcoma (Finnie, 1971, Frye, 1973, were not available to determine the duration of the presence Jacobson, eta l, 1981, Jacobson, et al, 1980, Goldberg, 1991). of the circulating blast-like cells and it is not known whether The possible role played by oncogenic viruses in neoplastic immaturity of cells is a valid criterion in snakes for classifica­ disease in reptiles has been discussed by others. Jacobson tion of leukemias as acute. It was not possible to determine described papillomas associated with infection by a papo- whether the leukemic blood profile originated from the bone

Volume 11, No. 4, 2001 Journal of Herpetological Medicine and Surgery 21 vavirus in side-necked turtles, Platemys platycephila, and a herpesvirus infection in European emerald , Lacerta viridis, (Jacobson, 1993). A “C”- type retrovirus present in cultured spleen cells from a Russell’s viper, Vipera russellia,1 with a splenic myxofibroma has been reported (Ziegal, 1969) j and a similar virus was identified in a corn snake, Elaphe gut-} tata, with a rhabdomyosarcoma (Lunger, et al, 1974).] Jacobson reported the occurrence of virus-like intranuclear^ inclusions in a California kingsnake with lymphosarcoma however the presence of viral particles could not be con-' firmed by transmission electron microscopy (Jacobson, et al, j 1980). Electron microscopy was not performed in this case. Venomous snakes present a unique safety challenge to the veterinarian in terms of anesthesia and handling for collection of diagnostic samples and performance of additional modali-; Downloaded from http://meridian.allenpress.com/jhms/article-pdf/11/4/19/2206983/1529-9651_11_4_19.pdf by guest on 29 September 2021 ties (radiographs, ultrasound) needed for a medical work-up. In this case, the use of an induction chamber, snake hook and syringe case to cover the fangs of the snake all served to mini­ mize exposure of personnel to accidental envenomation (for a Figure 4A. Markedly enlarged spleen surrounding the gall blad­ more complete review of anesthesia in venomous reptiles see der (arrow). Bar =1.0 cm. Heard, 2001).

Figure 4B. Splenic architecture is largely effaced by sheets of neoplastic lymphocytes. Bar = 50 pm.

Figure 4C. Same as Figure 4b. At higher magnification. Bar = 50 pm.

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