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Chronic T-cell lymphocytic leukemia in a black swan (Cygnus atratus): diagnosis, treatment, and pathology.

Abstract: An asymptomatic 14-year old, male black swan (Cygnus atratus) housed at a zoological institution was presented for routine preshipment examination. Hematologic findings indicated that the bird had a severe lymphocytic leukocytosis, consistent with chronic lymphocytic leukemia. Radiographs showed the presence of multiple soft tissue masses within the caudal coelomic cavity; ultrasound showed one mass to be an enlarged spleen, a cystic mass near the gonads, and a mass suspected to be associated with the ventriculus. Results of further antemortem diagnostics, including bone marrow aspiration, fine-needle aspirate cytology of the coelomic masses, and immunohistochemical staining confirmed T-cell leukemia with infiltration of the bone marrow and the spleen. The bird showed partial response to treatment with chlorambucil, lomustine, prednisone, L-asparaginase, and whole-body radiation, with neither evidence of adverse effects nor clinical signs of disease. Although the leukemia showed response, there was no evidence of remission at any point. The swan died 433 days after initial evaluation and initiation of therapy. Necropsy, histopathologic findings, and immunohistochemistry results confirmed extensive infiltration of multiple organs, including the liver, spleen, heart, lungs, and kidneys with neoplastic T-cell lymphocytes.

Key words: chronic lymphocytic leukemia, T-cell lymphocyte, chlorambucil, lomustine, radiation therapy, IHC, avian, black swan, Cygnus atratus

Case Report

A 14-year old, male black swan (Cygnus atratus), housed at a regional zoological institution, was presented to a referring veterinarian for a routine preshipment examination in preparation for transfer to another zoological facility. The bird had been reported as appearing healthy by the animal care staff and had an unremarkable medical history. On physical examination, no abnormal findings were recorded. A complete blood cell (CBC) count and biochemical profile were performed, and results of the biochemical profile were within reference intervals. The total white blood cell (WBC) count was 1 020 000 cells/[micro]L (reference interval mean, 13 410; minimum-maximum, 300048 700 cells/[micro]L), (1) and lymphocytes represented 98% of the WBC differential (999 600 cells/[micro]L; reference interval mean, 6006 cells/[micro]L; minimum-maximum, 245-30 200 cells/[micro]L). (1) A second CBC count was performed 2 days later and yielded similar results. The day after receipt of the second CBC count results, radiographs were performed, and a large, approximately 7.5-cm, well-demarcated, spherical soft-tissue opacity was identified in the right coelomic cavity. Nine days later, the swan was referred to the William R. Pritchard Veterinary Medical Teaching Hospital (VMTH), University of California, Davis, for further evaluation.

On presentation to the VMTH (day 10), the swan was bright and alert, with minor abnormal findings on physical examination. The bird's body weight was 8.5 kg and body condition score was 5/ 9. Contour feathers were absent along the cranial edge of the carpi bilaterally; the animal care staff had reported that the swan would attack caretakers by striking them repeatedly with its wings, and this was the suspected source of the feather loss. The left wing was pinioned at the proximal metacarpus. Blood samples were collected for a CBC count and plasma biochemical profile. Results of the CBC count showed severe lymphocytic leukocytosis (Fig 1), with 1 375 000 WBCs/ [micro]L, 1 360 000 lymphocytes/[micro]L, and 13 000 heterophils/[micro]L (reference interval mean, 6565 cells/[micro]L, minimum-maximum, 352-34 900 cells/[micro]L). (1) On blood smear evaluation, the presence of a large population of small, round, mature-appearing cells, approximately the size of a thrombocyte, with a very high nucleus to cytoplasm ratio was observed. These cells had mature, condensed chromatin, inapparent nucleoli, and a small volume of basophilic cytoplasm. The morphology and numbers of these lymphocytes were considered compatible with chronic lymphocytic leukemia (CLL). Biochemical profile findings were within reference intervals.

The swan was sedated with midazolam (1 mg/kg IM; Midazolam; Hospira, Inc, Lake Forest, IL, USA) (2) and butorphanol (1 mg/kg IM; Torbugesic, Fort Dodge Animal Health, Fort Dodge, IA, USA) (2) and was anesthetized with isoflurane in oxygen administered via face mask for whole-body radiographs and coelomic ultrasound. Radiographically, 3 coelomic soft tissue masses were identified. Based on anatomic location, one mass was suspected to be a markedly enlarged spleen (size not specified), the second a 7-cm X 9-cm soft-tissue mass near the gonads and adrenal glands, and the third a round soft tissue mass near and possibly contiguous with the ventriculus (size not specified). Coelomic ultrasound confirmed the presence of these 3 masses and identified the mass near the gonads as a fluid-filled cystic structure. Fine-needle aspiration of all 3 masses was performed, and samples were submitted for cytologic analysis. The cystic mass yielded approximately 60 mL of clear, viscous fluid, and a predominant population of small mature lymphocytes was observed on cytologic analysis. The aspirate sample of the apparent splenic mass was highly cellular; the sampled cells were consistent with either marked splenic infiltration of CLL or a primary splenic lymphoproliferative disease. Aspirates from the third mass yielded spindle cells and peripheral blood cells, and it was hypothesized that the suspected mass may have been associated with the ventriculus.

The next day, the swan was again anesthetized by the same protocol as the previous day, for a bone marrow aspirate. An 18-gauge bone marrow aspiration needle was used to collect a bone marrow sample via the left cnemial crest of the tibiotarsus. On cytologic evaluation of the sample, a monomorphic population of small, mature lymphocytes representing >30% of the total number of cells was identified. Additionally, erythroid hypoplasia was noted, but no abnormalities in the granulocytic and thrombocytic precursor lines were reported. The lymphocytes identified in the bone marrow sample were similar in morphology to the population of lymphocytes observed in the peripheral blood. These findings were considered diagnostic for CLL.

Based on reports of its use in treatment of this disease in other avian species, (3-5) chlorambucil (2 mg total dose PO q48h) (2) was recommended for treatment. Because this drug carries a risk of myelosuppression due to induced neutropenia, (6) it was recommended that a CBC count be performed every 2 weeks at discharge. The zoological institution also planned to monitor the swan's body weight each time a blood sample was collected.

On day 150, the swan was presented to the VMTH for reevaluation. The animal care staff reported it was still asymptomatic. Although the leukocytosis and lymphocytosis decreased at several points during treatment with chlorambucil, these changes were neither consistent nor persistent (Fig 1), and values remained severely increased above the upper limit of the reference interval. Physical examination findings were overall unchanged, with the exception of a mild bilateral pododermatitis and a 1-cm plantar lesion on the left intertarsal joint region. A blood sample was collected for a CBC count and biochemical profile. Biochemical findings were again within reference intervals, whereas lymphocytic leukocytosis was still present, with a total WBC count of 880 000 cells/[micro]L, with 871 000 lymphocytes/[micro]L and 880 heterophils/[micro]L (Fig 1). On blood smear cytologic analysis, a large population of small, mature lymphocytes was again identified, with characteristics similar to those noted on the prior analysis. Blood smear samples were stained for immunocytochemistry; results were positive for the CD3 marker and negative for the CD79a marker, supporting a diagnosis of CLL of T-cell origin.

The swan was anesthetized with the previous protocol for coelomic radiography, ultrasound, and bone marrow aspiration. Three intracoelomic soft tissue masses were again identified on radiographs and ultrasound: one most likely an enlarged spleen, one possibly an enlarged testicle, and one an unknown structure, suspected to be a de novo mass not clearly associated with a specific organ. Bone marrow aspiration analysis was repeated, this time sampling from the cnemial crest of the right tibiotarsus. Results confirmed the continued presence of small, mature lymphocytes within the bone marrow. Additionally, precursors of all 3 blood cell lineages (thrombocytes, granulocytes, erythrocytes) were decreased from the previous sample. Because chlorambucil had not produced the desired response, the chemotherapeutic protocol was changed. A single dose of L-asparaginase (400 IU/kg SQ; 3000 ILF total dose) was administered while the bird was hospitalized, and the bird was discharged with instructions to initiate lomustine (60 mg/[m.sup.2] PO q3wk; 20 mg). Upon return to the zoological facility, a CBC count was performed on a weekly basis for the first 3 weeks of treatment, then every 3 weeks for the next 3 months.

On day 219, prednisone (0.5 mg/kg PO q24h) was added to the chemotherapeutic protocol because of a lack of further improvement in the swan's leukemia. Because of the risk of immunosuppression and aspergillosis with steroid therapy, prophylactic antifungal therapy (itraconazole, 5 mg/kg PO q24h) was also initiated. These treatments were continued for 2 months, until the bird returned to the VMTH for evaluation.

On reevaluation on day 282, the swan's peripheral leukemia had shown little response to treatment with lomustine and prednisone, with leukocyte counts variably decreased from pretreatment values but not to the degree seen with chlorambucil therapy (Fig 1). However, the bird remained asymptomatic. Physical examination findings included mild pododermatitis, worse on the right foot, with the remainder of the findings unchanged from prior examinations. After consultation with a veterinary oncologist, palliative radiation therapy was offered as an alternative treatment. Subsequently, the swan was hospitalized and treated with whole-body radiation. A total dose of 2.0 Gy of external-beam radiotherapy, divided into 10 fractions of 20 cGy each, was delivered via a linear accelerator (Clinac 2100C; Varian Medical Systems Inc, Palo Alto, CA, USA), every 3-4 days (Mondays and Thursdays) over the course of 31 days. At the start of each week, a CBC count was performed. For each treatment, the bird was sedated with midazolam (1 mg/kg IM) and butorphanol (1 mg/kg IM) and mask-induced with isoflurane in oxygen. For the first treatment, the bird was orotracheally intubated for inhalant anesthesia because the length of the procedure was long to enable establishment of linear accelerator settings and positioning; all subsequent treatments were performed with inhalant anesthetic delivered via facemask. Weekly rechecks of the CBC count during radiation treatment showed significant improvement in the WBC count; however, those values were still markedly high (Fig 1). A bone marrow sample was obtained 24 days into radiation therapy (day 306) to monitor response. Compared with results of the previous aspirates, the numbers of granulocytic precursors were increased compared with neoplastic cells, although no erythrocytic precursors were identified. Radiographs were performed on the 28th day of radiation therapy (day 310) to reassess the coelomic masses; no changes in size or number of masses were observed.

The swan was discharged back to the zoological institution for further care. It was recommended to recheck a CBC count weekly for the first month and then less frequently if the leukemia appeared to be improving. If the swan's leukemia worsened, a second round of radiation therapy was planned, although a 1-year delay was recommended because of concerns about adverse effects from cumulative radiation exposure. (7) The swan remained asymptomatic for any outward clinical signs of CLL. At day 351, the total WBC count was decreased to 450 000 cells/[micro]L, with 441 000 lymphocytes/[micro]L; overall, the WBC count remained between 450 000 cells/[micro]L and 740 000 cells/[micro]L after whole-body radiation therapy (Fig 1).

On day 448, the animal care staff noted that the swan was exhibiting mild, occasional tremors. A CBC count and biochemical panel were performed (Fig 1); aside from the historical leukocytosis and lymphocytosis, the findings were considered clinically insignificant. Three days later, the tremors became much more prominent, and the bird was subsequently hospitalized. Based on clinical signs and history of severe lymphocytosis, hyperviscosity syndrome secondary to severe blood hypercellularity was suspected. (8,9) The swan was treated with cefovecin (8 mg/kg IM, Convenia, Zoetis, Florham Park, NJ, USA) and triamcinolone (0.5 mg/kg IM, Vetalog, Boehringer Ingelheim, St Joseph, MO, USA). The swan's clinical signs worsened, and the bird was found dead in its enclosure the next day, 6 days after developing the tremors and 450 days after initial diagnosis.

The carcass was submitted to the California Animal Health and Food Safety Laboratory System, Tulare branch, for necropsy. The swan was in good postmortem condition, was mildly emaciated, and weighed 7.73 kg. On gross examination, the liver appeared mildly enlarged and pale, and the left kidney and both testes had associated cysts. The spleen was not enlarged but was pale, as was the bone marrow. The third mass was not identified on gross necropsy. Tissue samples were collected and fixed in 10% neutral-buffered formalin. Samples were routinely processed, embedded in paraffin, cut in 4-[micro]m sections, stained with hematoxylin and eosin, and examined by bright-field microscopy.

On histologic evaluation of the bone marrow, multifocal, coalescing, and infiltrating foci of a uniform population of neoplastic lymphocytes with large, round to oval nuclei with scant eosinophilic cytoplasm (Fig 2A) were observed. Numbers of red blood cells and granulocytes were markedly reduced within the marrow. Severe periportal, perivascular, and sinusoidal infiltration with a similar population of neoplastic lymphocytes was found throughout the liver (Fig 3A). The normal splenic architecture was almost completely replaced by neoplastic lymphocytes (Fig 4A). Blood vessels, including glomeruli in the kidneys, and the heart and lungs had similar neoplastic cells. The cysts observed in the coelomic cavity were composed of thick, fibrous connective tissue and were attached to the capsule of the testes and kidney. There were no other significant lesions.

Formalin-fixed, paraffin-embedded sections of liver, spleen, kidneys, heart, lungs, and bone marrow were cut into 5-[micro]m sections and processed for immunohistochemical staining for lymphocyte markers. For positive controls, samples of the bursa of Fabricius (B cells) and thymus (T cells) from 3-week-old chickens were similarly prepared. The sections were deparaffinized in xylene substitute and rehydrated via graded ethanol, followed by blocking of endogenous peroxidases with 3% hydrogen peroxide solution. Heat-induced epitope retrieval was performed in a pressure cooker (Decloaking Chamber, Biocare Medical, Concord, CA, USA) at 95[degrees]C for 40 minutes with a modified citrate buffer formulation (Reveal, pH 6.0) for CD3, CD7, and TIA-1 or a high pH retrieval solution (Borg, pH 9.5-9.7) for CD4 + CD8 double-stain cocktail and PAX5. After applying a protein block (10 minutes), a standard immunohistochemistry (IHC) technique was performed with prediluted mouse monoclonal antibodies TIA-1 (clone TIA-1), PAX5 (clone BC-24), CD7 (clone LP15), CD4 (clone BC/1F6) + CD8 (SP16) rabbit monoclonal antibody (prediluted double stain cocktail), or rabbit polyclonal antibody CD3. All antibodies were obtained from the same source (Biocare). Antibodies were incubated at room temperature for 30 minutes. Detection was performed with an immunoglobulin G mouse antibody probe for 10 minutes, followed by a horseradish peroxidase polymer conjugate (MACH 4, Biocare) for 10 minutes (mouse antibodies) or 30 minutes (rabbit antibodies). A combination of a horseradish peroxidase polymer conjugate and an alkaline phosphatase polymer conjugate (MACH 2 Double Stain 2, Biocare) was also evaluated. Visualization was achieved with 3,3'-diaminobenzidine to produce a brown precipitate or Fast Red to achieve red staining at the antibody binding site. The slides were examined by microscopy and a semiquantitative staining scoring system was applied: 0, negative; +, mild; ++, moderate; and +++, severe. Neoplastic lymphocytes stained strongly positive with CD3 antibody in the bone marrow (Fig 2B), liver (Fig 3B), spleen (Fig 4B), heart, lungs, and kidneys (not shown), suggesting that the neoplastic lymphocytes were of T-cell origin (Table 1). Staining with TIA-1 was mild to moderate and negative for PAX5. Positive and negative controls used along with the tissues from the swan validated the immunohistochemical tests. Attempts to further classify the neoplastic T lymphocytes as either helper (CD4) or suppressor (CD8) cells with the rodent antibodies were not successful.

Discussion

In this report, we describe the diagnosis, treatment, and pathologic findings of chronic T-cell lymphocytic leukemia in a black swan. Although uncommonly reported in avian species, CLL has been previously documented in a green-winged macaw (Ara chloroptera), (5) a double yellow-headed Amazon parrot (Amazona ochrocephala oratrix), (3) and a European starling (Sturnus vulgaris) (4); leukemia has also been identified in an emu (Dromaius novaehollandiae), (10) a Pekin duck (Anas platyrhynchos domesticas), (11) and a saker falcon (Falco cherrug). (12) Lymphoproliferative neoplasias of retroviral (13) and herpesviral (14) etiology have been reported in domestic fowl, such as chickens, turkeys, and captive pheasants and quail. Unfortunately, neither virus isolation nor serology was performed in this case, so a viral etiology could not be determined.

Some of the previously reported cases were birds that were clinically ill at the time of diagnosis (lethargy, (4,11-12) inappetance, (3,10-12) weight loss, (10,12) and pelvic limb lameness (3)); others, like the swan in this report, did not show any overt clinical signs. (5) In many other species, animals are often asymptomatic at the time of diagnosis; however, lethargy, inappetance, and weight loss are commonly described. Lymphoma, a lymphoid neoplasia of solid organs, can become leukemic and may appear similar to CLL that has begun to infiltrate solid organs. (5,15) In other species, the degree of clinical signs tends to correlate with the degree of lymphocytosis, bone marrow involvement, and organ involvement. (16) Interestingly, leukocytosis was much more severe in this swan at diagnosis than in many of the birds in previous reports and yet the swan was clinically normal. The swan's lack of peripheral pancytopenia despite marked bone marrow involvement was thought to be a result of focal marrow involvement (with more normal marrow present elsewhere). Splenomegaly, commonly reported in dogs and cats with CLL, (16) was suspected radiographically and ultrasonographically in this bird, and splenic infiltration was identified at necropsy. Antemortem splenic involvement has not been identified in prior reports of CLL in birds but has been identified histologically postmortem (5,11,17); this may represent a less-common clinical feature of CLL in avian species.

Bone marrow involvement was identified in this swan, as it was in 2 other reported cases, by antemortem testing. (3,5) In all 3 cases, results of bone marrow cytology confirmed the diagnosis of CLL. Antemortem immunocytochemistry was performed for this swan and in 1 other case, (3) and postmortem IHC was performed in the third case (5); in all 3 birds, neoplastic cells stained positive for the CD3 T-cell marker. Mild-to-moderate TIA-1 immunoreactivity further supported the T-cell lymphocyte diagnosis; however, attempts to further classify the neoplastic T lymphocytes as either helper (CD4) or suppressor (CD8) cells with the rodent antibodies were not successful. The prognosis for the different phenotypes varies among species. In people, the T-cell form tends to be the most aggressive, (18,19) whereas in dogs, survival times are longer with T-cell CLL than with B-cell CLL. (18) Given the few avian cases reported to date, survival times of birds with T-cell lymphocytic leukemia are difficult to speculate.

Although treatment for CLL is not always indicated in asymptomatic individuals, (16) factors such as the lymphocyte count, organ involvement, cytopenia, lymphadenomegaly, fever, and secondary infections influence the decision whether to begin therapy. (18) In dogs, there is no consensus on how high the lymphocyte count should be before considering initiating chemotherapy; recommendations have ranged from initiating treatment at 60 000-100 000 cells/[micro]L. (16) In this report, this swan's severe degree of lymphocytosis and apparent splenic involvement led to the decision to initiate chemotherapy. Chlorambucil, an alkylating agent commonly used in treatment of CLL in people and dogs, has also been used at a dosage of 1-2 mg/kg q3-4 days to treat CLL in birds and 0.1-0.2 mg/kg (approximately 3-6 mg/[m.sup.2]) in dogs and cats. (6,16) In 2 reported cases of its use in birds, the patients showed some clinical response (4,5); however, a third bird did not show significant improvement in either clinical status or lymphocyte counts. (3) A fourth bird was treated with approximately 0.33 mg/kg chlorambucil twice weekly as part of a combination protocol with vincristine and prednisone. (11) Although clinical improvement in the bird's attitude and appetite were observed, the leukemia continued to worsen until the bird was euthanized 1 month after starting treatment. (11) Overall, large decreases in the WBC or lymphocyte counts have not been reported in avian species. Likewise, clinical response has been variable, ranging from improved activity and adequate quality of life to continued decline. This swan was treated with a dose of chlorambucil consistent with that used in prior case reports, and initially, both the WBC and lymphocyte counts decreased (1 375 000 cells/[micro]L to 880 000 cells/[micro]L, and 1 361 250 cells/[micro]L to 731 500 cells/[micro]L, respectively). However, lymphocyte counts can fluctuate with CLL, even without treatment, making it challenging to evaluate treatment efficacy; the perceived decrease in leukemic cells may have resulted from that fluctuation. Although subsequent hematologic testing showed no further improvement in cell counts, the bird remained asymptomatic. This is consistent with treatment goals in other species, where the aim is to control the disease but not fully eliminate it because true cures have not been reported. (16) A possible adverse effect of chlorambucil is thrombocytopenia and resulting potential coagulopathy; this was reported in a green-winged macaw and necessitated discontinuing treatment with that drug. (5) In canine and feline patients, reported side effects are myelosuppression and gastrointestinal toxicity (6); however, neither of these were observed in the swan. Studies on the ideal dosing and efficacy of chlorambucil are lacking in birds, so it is unclear whether the drug dosage used was appropriate for this species and this type of neoplasia; this may have led to subtherapeutic dosing and thus inadequate response. In some cases, a dose escalation is pursued if no effect is noted, and this might have been used for this swan. However, safety data on chlorambucil in birds is also limited, and higher doses may increase the risk of adverse effects. The dose used (approximately 0.24 mg/kg) is higher than that recommended for use in dogs and cats, (6,16) and because of the increased risk of adverse effects, such as the reported thrombocytopenia and coagulopathy in other species, (5) and limited evidence of benefit to a higher dose, (11) we did not increase the dose.

Because the swan appeared to stop responding to chlorambucil, it was discontinued in favor of lomustine, in hopes of achieving a better response. To our knowledge only 1 other avian species with CLL has been treated with cyclophosphamide and appeared to achieve disease control, and daily subcutaneous fluids and furosemide were used concurrently to encourage diuresis because of possible adverse effects associated with the urinary mucosa reported in other species. (5,20) This was not an ideal choice for this swan given its fractious nature. Potassium arsenite has been reported as a successful treatment for lymphoid leukemia in a saker falcon, but the systemic extent of that bird's neoplasia was not fully evaluated, and the reported follow-up period was only 1 month. (12) Newer medications used as first-line treatment in people, such as fludarabine, (21) were considered but were cost-prohibitive. Lomustine, or CCNU (1-[2-chloroethyl]3-cyclohexyl-l-nitrosurea), is another alkylating agent used in the treatment of other lymphoid neoplasms in veterinary patients (22-25) and in certain leukemias in human patients. (26,27) This drug was selected for use in this swan because of its demonstrated efficacy against other lymphoid neoplasias (22-25) as well as its ease of administration. Overall, treatment with lomustine did not reduce the lymphocyte counts, but numbers also did not increase. This response appears similar to that seen with chlorambucil in this swan and in other birds (5); whether the perceived lack of therapeutic response represents treatment failure or adequate disease control is difficult to evaluate. However, myelosuppression was not evident at this dosage of lomustine. As with chlorambucil, the paucity of data on effective dosing may have resulted in subtherapeutic dosing of lomustine for this swan. Prednisone was used as an adjunctive treatment when lomustine alone did not produce the desired reduction in the circulating leukemia. The dosage used (0.5 mg/kg q24h) was lower than those used in 2 other case reports (1 mg/kg q24h and 2.2 mg/kg q12h), (5,11) so it is unclear whether dosage affected the swan's outcome. However, regardless of other drugs included in the treatment protocols in other cases, including prednisone did not seem to help reduce the WBC count. (5,11,28) The use of L-asparaginase has been reported infrequently in treatment of avian neoplasia. It was used as part of a multidrug protocol for cutaneous lymphoma with leukosis in a Moluccan cockatoo (Cacatua moluccensis); the size of the solid tumor was reduced, but the bird remained leukemic despite treatment. (28) In another report, a great horned owl (Bubo virginianus), with a presumptive histiocytic sarcoma treated with lasparaginase, cyclophosphamide, and prednisone, developed severe bone marrow suppression. (29) The dose used was much higher (1650 U/kg once) that that used in the swan, (29) but it is unclear whether the higher dose of L-asparaginase or including cyclophosphamide led to the bone marrow suppression. Response to treatment may have been more pronounced with a higher dose of L-asparaginase, but this must be weighed against the risk of bone marrow suppression, which was not observed in this swan, even after adding prednisone to the chemotherapeutic protocol.

Total-body radiation was the final recommendation for treatment of the swan's CLL. This modality, often used in preparing bone marrow or stem cell transplants, has been proposed as a salvage therapy for relapsing multiple myeloma and acute myelogenous leukemia in people. (30,31) Because the lymphocyte count was not improving with alkylating agents, radiation was selected as a salvage attempt to reduce the swan's cell counts further. Radiation treatment was well-tolerated by this swan. Based on a prior study in ring-necked parakeets (Psittacula krameri), (32) no severe acute adverse effects of the radiation were expected, and none were identified after the end of therapy. Radiation treatment was somewhat effective in this swan because the WBC count dropped almost 400 000 cells/[micro]L over the course of treatment and remained lower than preradiation baseline values. However, the percentage of WBCs identified as lymphocytes never dropped below 94%.

Reported survival times for CLL in avian species are usually short, up to approximately 2 months after starting treatment. (3,10,11) However, those birds that showed lasting response to treatment had survival times of at least 6 months (including this swan) (4,10) to more than a year. Interestingly, 2 birds with CLL with documented survival times of more than 1 year were both treated with chlorambucil, cyclophosphamide, and prednisone. (5) To our knowledge, lomustine use has not been reported in avian species, and only one case of L-asparaginase being used has been reported, with a survival time of 8 weeks. (29) Future descriptions of the use of these drugs will be valuable in assessing their efficacy in treatment of avian leukemias.

Progressively worsening neurologic signs were observed in the days preceding the swan's death. In people with hyperleukocytosis secondary to leukemia and other myeloproliferative neoplasias, neurologic signs, such as hearing loss, (33) vertigo, (8) ataxia, (9) headache, (9) nystagmus, (9) and other neurologic signs, have been described, secondary to hyperviscosity syndrome. (8,9,33) The many neoplastic cells in the bloodstream increases blood viscosity, hindering blood flow and leading to tissue hypoxia. (4,8,9,33) Suspected hyperviscosity syndrome has also been described in a starling diagnosed with CLL (4) and may explain this swan's tremors.

Because the peripheral lymphocytosis never fully resolved in this swan, necropsy findings of disseminated neoplastic lymphocytes in multiple organs were not unexpected. In 2 case reports of birds treated for T-cell CLL, neoplastic cells were identified in multiple organs and tissues despite treatment with chlorambucil or cyclophosphamide. (3,5) The tissue distribution of neoplastic cells in this swan was similar to that reported in other avian species, with bone marrow, liver, intestines, and spleen similarly affected. (3,5,11,17) This swan had more-disseminated disease than reported in other birds, with the adrenal glands and aorta also infiltrated with neoplastic cells. These findings seem to suggest that in avian species, liver and bone marrow may be target organs for biopsy if this disease is suspected. Similarly, in mammalian species, bone marrow and sampling of any enlarged organ is performed. (16) In all 3 birds, the neoplastic cells (both antemortem and postmortem) stained strongly for the CD3 marker, indicating a T-cell CLL. This staining should be considered for blood samples, bone marrow aspirates, or other organ biopsy samples from leukemic birds.

Although CLL appears to be an uncommon diagnosis in birds, it should be considered as a differential in a bird with vague clinical signs or neurologic signs (attributable to hyperviscosity syndrome). However, CLL may also be found as an incidental finding in an otherwise asymptomatic bird. If treatment is considered, chlorambucil may reduce, but not eliminate, circulating neoplastic lymphocytes. Although no adverse effects resulting from use of this drug were observed, prior reports suggest that the patient must be monitored carefully for thrombocytopenia. This swan's response to lomustine and prednisone did not appear significant, but whether this indicates lack of response or prevention of disease progression is unclear; further trials may prove its efficacy. Whole-body radiation resulted in a pronounced decrease in lymphocyte counts in this swan and may be useful as a rescue therapy should chemotherapeutic drugs fail in other cases.

Acknowledgments: We thank David Tacha and Margaret Lobo of Biocare Medical (Concord, CA, USA) for performing IHC, and the staff at CAHFS, Tulare, for their technical assistance.

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Kristin M. Sinclair, DVM, Dipl ABVP (Avian), Michelle G. Hawkins, YMD, Dipl ABVP (Avian), Lewis Wright, DVM, Richard P. Chin, DVM, MPVM, Dipl ACPV, Sean D. Owens, DVM, Dipl ACVP, David Sanchez-Migallon Guzman, LV, MS, Dipl ECZM (Avian, Small Mammal), Dipl ACZM, Michael S. Kent, DVM, Dipl ACVIM, Dipl ACVR, and H. L. Shivaprasad, BVSc, PhD, Dipl ACPV

From the William R. Pritchard Veterinary Medical Teaching Hospital (Sinclair) and the Departments of Veterinary Medicine and Epidemiology (Guzman, Hawkins), Pathology, Microbiology, and Immunology (Owens), and Surgical and Radiological Sciences (Kent), School of Veterinary Medicine, University of California, Davis, One Shields Ave, Davis, CA 95616, USA; the Fresno Chaffee Zoo, 894 W Belmont Ave, Fresno, CA 93728, USA (Wright), and the California Animal Health and Food Safety Laboratory System, Tulare Branch, University of California, Davis, 18830 Rd 112, Tulare, CA 93274, USA (Chin, Shivaprasad).

Table 1. Results of immunohistochemical staining of tissue
samples from a black swan with chronic lymphocytic leukemia and
from control samples.

                               Chicken              Chicken bursa
Antibodies    Swan   (with chronic inflammation)   (B-cell control)

CD3           +++                ++                       +
TIA-1          +                 ++                       0
PAX5           0                  0                      +++
CD4            0                  0                       0
CD7            0                  0                       0
CD8            0                  0                       0

               Chicken thymus    Negative controls
Antibodies    (T-cell control)      (antibody)

CD3                 +++                  0
TIA-1                ++                  0
PAX5                 0                   0
CD4                  0                   0
CD7                  0                   0
CD8                  0                   0

Abbreviations: CD3 indicates T-cell coreceptor: TIA-1, cytolytic
T cells and natural killer cells; PAX5, B cells; CD4, T-helper
cells; CD7, T lymphocytes; CD8, T suppressor cells.
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Author:Sinclair, Kristin M.; Hawkins, Michelle G.; Wright, Lewis; Chin, Richard P.; Owens, Sean D.; Guzman,
Publication:Journal of Avian Medicine and Surgery
Article Type:Report
Date:Dec 1, 2015
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