Long-term treatment of chronic lymphocytic leukemia in a green-winged macaw (Ara chloroptera).
Article Type: Clinical report
Subject: Cyclophosphamide (Health aspects)
Cyclophosphamide (Dosage and administration)
Cyclophosphamide (Research)
Prednisone (Health aspects)
Prednisone (Dosage and administration)
Prednisone (Research)
Chemotherapy (Research)
Chemotherapy (Health aspects)
Chronic lymphocytic leukemia (Care and treatment)
Chronic lymphocytic leukemia (Drug therapy)
Chronic lymphocytic leukemia (Research)
Chronic lymphocytic leukemia (Causes of)
Macaws (Health aspects)
Macaws (Care and treatment)
Macaws (Research)
Chlorambucil (Dosage and administration)
Chlorambucil (Health aspects)
Chlorambucil (Research)
Cancer (Chemotherapy)
Cancer (Research)
Cancer (Health aspects)
Authors: Hammond, Elizabeth E.
Guzman, David Sanchez-Migallon
Garner, Michael M.
Mauldin, Glenna
Martinez-Jimenez, David
Aguilar, Roberto F.
Pub Date: 12/01/2010
Publication: Name: Journal of Avian Medicine and Surgery Publisher: Association of Avian Veterinarians Audience: Academic Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2010 Association of Avian Veterinarians ISSN: 1082-6742
Issue: Date: Dec, 2010 Source Volume: 24 Source Issue: 4
Topic: Event Code: 310 Science & research
Geographic: Geographic Scope: United States Geographic Code: 1USA United States
Accession Number: 252005077
Full Text: Abstract. A 32-year-old green-winged macaw (Ara chloroptera) was diagnosed with chronic lymphocytic leukemia based on progressive lymphocytosis and the presence of a monomorphic population of well-differentiated lymphocytes in the bone marrow of a clinically normal bird. Chemotherapy was initiated because of rapidly increasing peripheral lymphocyte counts. In addition to oral prednisone (1 mg/kg once daily), oral chlorambucil (1 mg/kg twice weekly) was initiated but was discontinued after 6 weeks because of thrombocytopenia. The leukocyte count was stabilized for 29 weeks with the concurrent use of oral cyclophosphamide (5 mg/kg 4 d/wk) and daily prednisone, and the bird exhibited a good quality of life. The bird died shortly after the chemotherapy was inadvertently discontinued. The neoplastic cells from this macaw stained positive for CD-3 antibody and negative for Bla.36, suggesting the leukemia was of T-cell origin. This is the first report of long-term treatment of a macaw with cyclophosphamide and documents thrombocytopenia in a macaw secondary to chlorambucil, treatment.

Key words: chronic lymphocytic leukemia, thrombocytopenia, T cell, chemotherapy, chlorambucil, cyclophosphamide, avian, green-winged macaw, Ara chloroptera

Clinical Report

A 32-year-old, male green-winged macaw (Ara chloroptera) was presented for annual examination. The bird weighed 1.08 kg and appeared healthy. Anesthesia was induced and maintained with isoflurane (2% isoflurane administered via face mask), and a blood sample was taken from the basilic vein for a complete blood cell count (CBC) and plasma biochemical analysis. Results of the CBC revealed leukocytosis (41 000 cells/[micro]L; reference range, 6000-14 000 cells/[micro]L) with lymphocytosis (33 210 cells/[micro]L; reference range, 2700-9800 cells/[micro]L; 81% of leukocytes, reference range, 45%-70%) and normal heterophil count (7790 cells/[micro]L; reference range, 2700-9800 cells/[micro]L; 19% of leukocytes, reference range, 45%-70%). (1) The only abnormal biochemical result was a mild increase of creatine phosphokinase activity (2198 IU/L, reference range 100-1500 IU/L), (1) which was attributed to muscle trauma from handling. Antibody testing for Chlamydophila psittaci IgM by elementary body agglutination was negative (< 1:10). Avian polyomavirus and psittacine beak and feather disease virus DNA test results were also negative. The bird recovered uneventfully and was returned to its enclosure for monitoring.

Twenty-nine weeks after initial health examination the macaw was anesthetized as before for physical examination, repeat CBC, and whole-body radiographs. The bird weighed 1.15 kg, and results of the physical examination were unremarkable. Ventrodorsal and lateral radiographic examination revealed mild diffuse increased opacity in both lungs. The only abnormality on the CBC was severe leukocytosis (65 000 cells/[micro]L) with lymphocytosis (59150 cells/[micro]L, 91% of leukocytes). Evaluation of the blood smear was not recorded.

Three weeks after the severe leukocytosis was identified, the bird was manually restrained for blood collection. Results of the CBC revealed a notably increased total white blood cell (WBC) count of 250000 cells/[micro]L, with 240000 cells/[micro]L comprising a homogeneous population of lymphocytes. Results of the biochemical analysis showed a high concentration of total protein (5.8 g/dL, reference range 3.0-5.0 g/dL). (1) Results of plasma protein electrophoresis revealed decreased albumin:globulin (A/G) ratio (0.58, reference range 0.8-2.6), increased beta globulin concentration (0.89 g/dL, reference range 0.38-0.54 g/dL), and a marked hypergammaglobulinemia (2.21 g/dL, reference range 0.21-0.55 g/dL), (2) suggesting a significant immune response. The test result for Chlamydophila psittaci (elementary body agglutination) was negative, whereas the Aspergillus titer (enzyme-linked immunosorbent assay) was positive (1.056, reference range 0.000-0.330). (3) Results of direct fecal examination, fecal flotation (zinc sulfate-saturated solution), and acid-fast staining were negative for parasites and acid-fast organisms.

Three days later, bone marrow aspiration from the right tibiotarsus was performed with the macaw under general anesthesia with isoflurane. Cytologic examination of bone marrow aspirates stained with modified Wright-Giemsa technique revealed a preponderance of small, well-differentiated lymphocytes, few plasma cells, and very low numbers of myeloid and erythroid precursors. The myeloid to erythroid ratio was 5 : 1, but only 25 hematopoietic cells could be found in the cytologic preparations. The low hematopoietic cell count was attributed to myelophthisis. These findings were consistent with a well-differentiated lymphoid malignancy in the bone marrow and supported a diagnosis of chronic lymphocytic leukemia (CLL).

Based on the elevated Aspergillus titer, aspergillosis was suspected, and the bird was started on itraconazole (5 mg/kg PO q12h for 5 days, then q24h; Sporanox, Ortho-McNeil-Janssen Pharmaceuticals, Titusville, NJ, USA). Because of the risk of immunosuppression, the bird was also started on enrofloxacin (10 mg/kg PO q12h; Baytril 2.27%, Bayer HealthCare LLC, Shawnee Mission, KS, USA) to cover the possibility of a bacterial infection.

Thirty-four weeks after presentation and 7 weeks after the severe leukocytosis was identified, the macaw was transported to the Louisiana State University Veterinary Teaching Hospital for coelioscopy to evaluate the bird for neoplastic or fungal masses. Anesthesia was induced with isoflurane and maintained with isoflurane 2% administered by endotracheal tube. Blood was collected from the right jugular vein for a CBC and plasma biochemical analysis. Results revealed considerable worsening of the leukocytosis (total WBC, 489000 cells/[micro]L), primarily due to lymphocytosis (440 000 cells/[micro]L). Mild anemia (packed cell volume [PCV] 36%, reference range, 45%-55%) was also present. (1) A coelomic examination was performed with a 2.7-mm rigid endoscope (Karl Storz Veterinary Endoscopy America Inc, Goleta, CA, USA), on both cranial and caudal left thoracic air sacs and the left abdominal air sac. Coelioscopy revealed pulmonary petechiation and ecchymosis on the visceral side of the left lung. The air sac membranes were slightly opaque, but no overt fungal plaques were observed. An irregularly shaped, pale, highly vascular mass (size not recorded) was visible near the caudo-ventral edge of the left lung and adjacent to the proventriculus. Also, a large vascular structure (size not recorded) was observed between the cranial and medial parts of the left kidney. Because of the prominent vasculature associated with the masses, biopsy was not attempted because of the risk of severe hemorrhage. Although the kidney and liver were grossly considered normal, biopsy samples were obtained from both organs. Tracheoscopy revealed no abnormalities, but a deep tracheal swab was taken for fungal culture, which was negative. The bird recovered uneventfully. Enrofloxacin was continued orally, and piroxicam (0.75 mg/kg PO q12h for 3d; Feldene, Pfizer Inc, New York, NY, USA) was initiated for its analgesic and antitumor properties. (4)

Based on the bird's peripheral lymphocytosis, the large population of small, well-differentiated lymphocytes in the bone marrow, and the absence of clinical signs, CLL was diagnosed. Chemotherapy was started because of the rapid and dramatic increase in peripheral lymphocyte counts, as well as the development of associated cytopenias (anemia). Histologic examination of the hepatic and renal biopsy samples revealed mononuclear cells whose exact lineage could not be determined but which were suggestive of a lymphoid neoplasm. Because of the positive Aspergillus titers, nebulization with clotrimazole (3 mL q24h; clotrimazole topical solution 1% USP, Teva Pharmaceuticals USA, Sellersville, PA, USA) was initiated 4 days after endoscopy. The bird was exposed to the nebulized clotrimazole daily for 1 hour total.

One week after endoscopy and before initiating chemotherapy, the bird was anesthetized as before for blood sample collection. Lymphocytic (276 000 cells/[micro]L, 92% of leukocytes) leukocytosis (total WBC 300000 cells/[micro]L) and mild anemia (PCV 38%; red blood cell [RBC] count, 2.05 x [10.sup.6]/[micro]L; reference range, 2.5-4.5 x [10.sup.6] cells/[micro]L) were still present. (1) The biochemical values were unremarkable, and the protein electrophoresis values indicated markedly decreased A/G ratio of 0.42 and increased gamma globulin of 2.12 g/dL. The Aspergillus titer had decreased (0.420) but was still above normal. The next day (35 weeks after initial presentation), chemotherapy was initiated with chlorambucil (1 mg/kg PO 2 times weekly, at 3- and 4-day intervals; Leukeran, GlaxoSmithKline, Research Triangle Park, NC, USA, compounded into a 2 mg/mL suspension by Carr Drugs, New Orleans, LA, USA) and prednisone (1 mg/kg PO q24h). Latex gloves were worn when handling chlorambucil to prevent inadvertent toxicity to staff.

One week later, a blood sample was obtained from the basilic vein for repeat CBC and viral testing to rule out viral disease as the underlying cause of the bird's lymphoproliferative disease. Clinically significant values included increasing lymphocyte count (405000 cells/[micro]L, 90% of leukocytes) and heterophilia (45000 cells/[micro]L, 10% of leukocytes), a normal PCV (45%) but low total RBC count (2.39 x [10.sup.6]/[micro]L). The Aspergillus titer remained slightly positive (0.409). Results for polyomavirus serologic testing, psittacine beak and feather disease virus polymerase chain reaction (PCR) assay, and reticuloendothelial virus PCR assay were negative. Herpesvirus was not detected in the blood by PCR using primers specific for psittacine herpesvirus or degenerate primers. Results of physical examination were unremarkable, and chemotherapy was continued.

Thirty-eight weeks after initial presentation and 3 weeks after initiation of chlorambucil treatment, the bird was anesthetized for blood sample collection for a repeat CBC. A lower WBC count (275000 cells/[micro]L) was noted, suggesting a response to the current treatment. However, lymphocytosis (253000 cells/[micro]L, 92% of leukocytes), heterophilia (16 500 cells/[micro]L, 6% of leukocytes), and anemia (PCV 39%; RBC count 2.15 x 106 cells/[micro]L) persisted.

Approximately 6 weeks after initiation of the chlorambucil-prednisone treatment, the bird was restrained for routine phlebotomy. Subjectively, hemostasis after phlebotomy was prolonged, and thrombocyte count was low at 14000 cells/[micro]L (reference range, 20000-30 000 cells/[micro]L). (1,5) Based on the suspicion of chlorambucil-induced thrombocytopenia, this drug was discontinued. Prednisone was continued. The total WBC count at this time was 300 000 cells/[micro]L with lymphocytosis (294 000 cells/[micro]L, 98% of leukocytes), and a mild anemia (PCV 40%; RBC count 2.25 x [10.sup.6] cells/ [micro]L) was still present. Results of the biochemical analysis were within reference ranges. Although still high, the gamma globulin levels were lower (1.32 g/dL), and the A/G ratio of 0.75 was just below the reference range.

One week after cessation of the chlorambucil, the bird was anesthetized as before for phlebotomy and bone marrow aspiration to evaluate the effect of treatment on the bone marrow. A noticeable difference in hemostasis was observed, and the blood clotted immediately after digital pressure was applied over the venipuncture site. Although a thrombocyte count was not performed because of laboratory error, adequate numbers of thrombocytes were observed by the laboratory. The WBC count was 190 000 cells/[micro]L. Results of evaluation of the bone marrow aspirate sample were similar to the previous sample, revealing well-differentiated lymphoid cells suggestive of CLL.

Sixteen days after cessation of the chlorambucil, thrombocytes were considered adequate (35 000 cells/[micro]L) and were similar in value to a thrombocyte count in a healthy macaw at the same facility. The WBC count was 250 000 cells/ [micro]L with 96% lymphocytes and 4% heterophils. At approximately 46 weeks after presentation, cyclophosphamide (5 mg/kg PO q24h for 4 days, repeat in 21 days; Cytoxan, Bristol-Myers Squibb Company, Princeton, NJ, USA, compounded into a 5 mg/mL suspension by Carr Drugs) was added to the prednisone treatment; latex gloves were worn when handling the chemotherapeutic agent. Because cyclophosphamide has been shown to cause hemorrhagic cystitis in mammals, the bird was given lactated Ringer's solution (25 mL SC q24h for 4 days) and furosemide (2 mg/ kg SC q24h for 4 days) with the cyclophosphamide treatment to ensure proper diuresis. The daily prednisone, itraconazole, and nebulized clotrimazole were continued.

Blood samples collected weekly over the next 3 weeks showed a mild increase and then stabilization of the WBC count (275 000 cells/[micro]L, 325000 cells/[micro]L and 325000 cells/[micro]L; Fig 1). Three weeks after the initial cyclophosphamide dose, it was reinitiated q24h for 4 days and stopped for 3 days. This cycle of 4 days of cyclophosphamide, 3 days without cyclophosphamide was continued weekly. White blood cell counts were performed weekly, and biochemical analyses were done monthly. During this time, the White blood cell count never exceeded 375 000 cells/[micro]L (Fig 1), and thrombocyte counts were normal. The bird tolerated the treatment well, maintained its weight, and appeared to have a good quality of life.

[FIGURE 1 OMITTED]

Seventy-five weeks after initial presentation and 29 weeks after the initiation of cyclophosphamide, Hurricane Katrina disrupted routine husbandry and medical care at the zoological facility. The bird's medications were discontinued for 5 weeks, at which point a physical examination was performed. Despite being alert and responsive, the bird exhibited mild paresis of the left leg. Palpation of limbs and full body radiographs revealed no significant findings, and the paresis was attributed to soft tissue trauma. Results of a CBC showed the WBC count was extremely elevated (1 121 000 cells/[micro]L) with 98.5% lymphocytes and anemia (PCV 28%). Five weeks after cessation of the bird's medications, oral prednisone, cyclophosphamide, itraconazole, and nebulized clotrimazole were restarted. Results of a CBC 1 week after the medications were restarted revealed a less pronounced lymphocytic leukocytosis (400 000 cells/[micro]L, 95% lymphocytes) and anemia (PCV 34%; RBC count 1.80 x [10.sup.6] cells/[micro]L). A biochemical analysis was not performed.

Two weeks after initiation of the medications, the bird appeared lethargic and weak with fluffed feathers and was admitted to the hospital. Its weight was 0.9 kg, and the macaw was in thin body condition, appeared dehydrated, and had poor feather quality. The liver was palpably enlarged. The bird was perching but unable to balance if moved. A blood sample obtained from the right basilic vein revealed severe anemia (PCV 9%) and hyperglycemia (glucose, 495 mg/dL; reference range, 200-350 mg/dL). (1) A CBC was not available due to laboratory error. Treatment with cyclophosphamide, prednisone, itraconazole, and nebulized clotrimazole was continued. The bird was started on marbofloxacin (5 mg/kg PO q24h; Zeniquen, Pfizer Animal Health, Exton, PA, USA) to treat a possible bacterial infection due to immunosuppression from the CLL, and lactated Ringer's solution (25 mL SC once) and enteral nutrition (30 mL PO once; Emeraid Critical Care, Lafeber Company, Cornell, IL, USA) were also administered. The bird died before additional diagnostic tests could be pursued.

Necropsy revealed thickening of the caudal thoracic and abdominal air sac membranes. The liver was enlarged and had small, disseminated white foci. The great vessels at the base of the heart were enlarged. There was no evidence of the masses previously observed during coelioscopy. Histologically, the lymphoid malignancy was distributed throughout the bone marrow, liver, pancreas, intestines, lung, proventriculus, spleen, and air sacs. Solid foci of the neoplasm comprised sheets of round ceils with small hyperchromatic nuclei containing small nucleoli. Rare mitotic figures were present. The neoplastic cells were occasionally admixed with low numbers of well-differentiated plasma cells and Mott cells and scattered histiocytes. The neoplastic cells stained uniformly and intensely positive for CD3, a T-cell marker. The neoplastic cells stained uniformly negative for Bla.36, a B-cell marker, although this marker did stain the plasma cells and histiocytes in and around the neoplastic population (Fig 2). In the small intestine, a focus of pseudomembrane formation and ulceration associated with large numbers of large bacilli was present in the mucosa adjacent to a focus of transmural ulceration and neoplastic infiltration. The aorta had a partially occlusive and recanalized thrombus. The heart had some mild interstitial fibrosis in the ventricles and mild left ventricular myocardial necrosis.

Discussion

Lymphoid neoplasia is the most common form of hemolymphatic neoplasia reported in avian species. (6-8) This type of neoplasia is commonly diagnosed in poultry and has occasionally been reported in mostly captive and companion birds of the orders Passeriformes, Psittaciformes, and Columbiformes, (6,7,9) and in a small number of raptors. (10-13) In psittacine birds, lymphoproliferative disorders are not common in any species but have been described in budgerigars (Melopsittacus undulatus), cockatoos (Cacatua species), an Amazon parrot (Amazona ochrocephala oratrix), and macaws (Ara species). (5,7,14-17) Lymphoproliferative tumors may manifest in the skin, (8,14,18) although disseminated visceral disease (lymphoma) has also been reported. (17,19,20) In most of these cases, treatment was not initiated or was unsuccessful. To our knowledge, this is the first reported case of long-term treatment of CLL in a macaw.

The clinical diagnosis of CLL in this 32-year-old male green-winged macaw was made based on the abnormal hematologic results with progressively increasing lymphocytic leukocytosis and anemia and the presence of a monomorphic population of well-differentiated lymphocytes in the bone marrow aspirate in a clinically normal bird. These diagnostic criteria are extrapolated from the small animal literature. Chronic lymphocytic leukemia is often diagnosed in asymptomatic domestic mammals in which absolute peripheral lymphocytosis is the major diagnostic abnormality and is associated with significant infiltration of the bone marrow by well-differentiated lymphocytes. (21) The bird in this report had a severe leukocytosis; the WBC count ranged from 20 000 to 1 121 000 cells/[micro]L with over 90% lymphocytes most of the time. There is one report of a saker falcon (Falco cherrug) with lymphoid leukemia in which the total WBC count was estimated at 1 700 000 to 1 800 000 cells/[micro]L, with 99% lymphocytes. (12) Other hematologic abnormalities in domestic animals with CLL include anemia and hyperglobulinemia, both of which were present in the green-winged macaw with CLL. The bird in this case had a significant hypergammaglobulinemia. Monoclonal gammopathies have been reported in dogs with CLL in which serum is evaluated by protein electrophoresis. (21) Monitoring results of protein electrophoresis can also aid in evaluating response to treatment.

Lymphoid leukemia by definition is a tumor of peripheral blood and bone marrow, as was the case in the macaw reported here. (6) This is in contrast to lymphoma, which develops as a solid tumor in multiple tissues and can involve the bone marrow. (6) Diagnosis of CLL in birds can be challenging, particularly because this syndrome is poorly characterized in the avian patient. The advanced stage of CLL at the time of diagnosis may make it difficult to identify the origin of the neoplasia in birds. By the time the disease is diagnosed, the neoplasia may have disseminated to multiple organs including coelomic viscera and may confound the determination of its origin. (6,10)

[FIGURE 2 OMITTED]

The positive CD3 immunohistochemical staining characteristics of the neoplasm in this case suggest it was of T-cell origin. (19,22) The neoplastic cells stained negative for Bla.36, a B-cell marker that has also been shown to detect histiocytic cells in numerous species. (8,18,23)

Avian lymphoproliferative disorders have been associated with herpesvirus (Marek's disease) and retroviruses (avian leukosis virus and reticuloendotheliosis virus), (10,18) Some lymphoid neoplasms in psittacine birds are suspected to be caused by viruses because of the resemblance to those syndromes caused by RNA viruses in domestic chickens. However, there is currently no research proving this, (6,8) and viral particles have not yet been demonstrated. (5,14,18) Viral infection was not identified in this case based on negative serologic and PCR testing for known psittacine viruses. (24,25)

Criteria for treatment of CLL include clinical signs of disease, degree of increase in the peripheral lymphocyte count, evidence of organ involvement, associated hematologic abnormalities such as anemia, and rapid lymphocyte doubling time in less than 6 months. (21) Chemotherapy was initiated in this case because of the dramatic lymphocytic leukocytosis, the lymphocyte doubling time over a short time period, anemia, and bone marrow involvement. In addition, lymphoid neoplasia often leads to severe clinical disease in pet birds over time. (6) The goal of CLL therapy in humans and small animals is to control the disease and spare the bone marrow, as complete cures are rare. (21,26) It may take more than a month for the hematologic and clinical abnormalities to resolve. However, long survival times (more than 2 years in dogs) may be seen in animals with CLL even without treatment. (21) Unfortunately, staging and grading schemes to direct treatment have not been developed for avian species. (6) Dosages and regimens of chemotherapeutic agents have been published with different results and are usually derived from current small animal or human protocols, although most are based on an alkylating agent paired with prednisone. (5,8,11,12,17,27)

Chlorambucil was selected for its efficacy in treating CLLs in humans, (26) cats, (21) dogs, (21) and birds, (5,8) with relatively few reported toxicities. Chlorambucil is an alkylating agent with immunosuppressant and antineoplastic properties. In one report, a pekin duck (Anas platyrhynchos domesticus) with CLL was treated with chlorambucil in combination with other chemotherapeutic agents, but the bird died soon after treatment was initiated. (28) Thrombocytopenia was observed in the duck during chemotherapy, but the cause was not determined. A dosage of 2 mg/kg twice weekly has been used to successfully treat cutaneous lymphosarcoma in an umbrella cockatoo (Cacatua alba). (8) A dosage of 2 mg/kg chlorambucil once every 14 days for 3 treatments was used to successfully treat a blue and gold macaw (Ara ararauna) with cutaneous pseudolymphoma. (5) The authors reported normal thrombocyte counts during the treatment period. Treatment with chlorambucil at a dosage of 1 mg/kg twice weekly in this greenwinged macaw was discontinued after 6 weeks because of prolonged hemostasis and thrombocytopenia. Bone marrow suppression, thrombocytopenia, and hepatotoxicity have been associated with chlorambucil administration in other species. (28) In this case, no evidence of hepatotoxicity was observed, and the biochemical results remained unremarkable. Birds with lymphoid leukemia may have low thrombocyte counts, (6) although this macaw's thrombocyte count returned to normal after stopping the drug, suggesting chlorambucil may have contributed to thrombocytopenia in this case. Because of the short duration of treatment, the efficacy of the drug in this case is unknown.

Cyclophosphamide, a derivative of nitrogen mustard, was added to the protocol treatment in lieu of chlorambucil based on small animal protocols. (21) Cyclophosphamide was chosen because it is platelet-sparing, readily available, and cost-effective, and because, like chlorambucil, it is an alkylating agent. (29) In addition, it is available as an oral formulation, which was important in a bird that was not a candidate for repeated intravenous treatment. The bird tolerated the drug well. A potential side effect of cyclophosphamide in small animals is hemorrhagic cystitis, which is thought to be due to a direct toxic effect of cyclophosphamide's metabolite, acrolein, on the urinary tract mucosa. (30) In dogs receiving cyclophosphamide, concurrent furosemide administration reduced the risk of sterile hemorrhagic cystitis. (30) Because of this potential side effect, the bird reported here was treated prophylactically with furosemide and sub-cutaneous fluids. No adverse urinary effects were seen in this bird with cyclophosphamide treatment, and the furosemide and fluid treatment regimen frequency was reduced to every other cyclophosphamide treatment because of contusions noted from multiple injections. Anatomic differences between avian and mammalian urinary tracts or differences in metabolism may make the urinary side effects of cyclophosphamide less likely in avian patients.

Gastrointestinal toxicity was suspected in a report of intraosseous cyclophosphamide administration in a Moluccan cockatoo (Cacatua moluccensis). (17) The green-winged macaw in this case exhibited an episode of regurgitation 1 day after initiation of oral cyclophosphamide, but no additional gastrointestinal side effects were observed. Cyclophosphamide treatment may have helped stabilize the WBC count in this macaw. The bird's precipitous demise after cessation of the chemotherapy for 5 weeks and the dramatic decrease in peripheral lymphocyte count that was observed shortly before death when cyclophosphamide therapy was restarted suggest that the chemotherapy prevented progression of the leukemia.

Corticosteroids are commonly used in conjunction with an alkylating agent to treat CLL because of their immunosuppressive and lymphocytolytic properties. (21) The dosage of prednisone was similar to that used in small animal chemotherapeutic protocols. (21) Adding additional chemotherapeutic drugs, changing drugs, and increasing doses would have been other options for intensifying treatment for the bird had it survived.

The presumptive concurrent pulmonary aspergillosis based on elevated titers was not confirmed on tracheal cultures or postmortem examination. Aspergillosis can be challenging to diagnose in the avian patient. False positive results can occur with serologic testing. (31-33) Protein electrophoresis can aid in the diagnosis of aspergillosis. The macaw had an increase in gamma globulin concentrations and a reduced A/G ratio, both of which have been seen in birds with aspergillosis. (32) Regardless, the patient was considered susceptible to developing fulminant aspergillosis due to potential immunosuppression from the CLL (6,10,34) and chemotherapy. In birds, lymphoid tumors may cause immunosuppression by interfering with cell-mediated and humoral immunity. (10)

This bird likely died of complications from advanced CLL, secondary bacterial enteritis, and associated intestinal perforation. The bacterial infection was likely precipitated by the intestinal neoplastic infiltrate and immunosuppression. The anemia was most likely caused by complete effacement of the bone marrow by the neoplastic cells, although the chemotherapy and undocumented hemorrhage may have been contributing factors. In future cases with avian patients on immunosuppressant drugs, close monitoring for evidence of secondary bacterial or fungal infections is warranted.

This case contributes to the relatively few reports of CLL and long-term chemotherapy in birds. The green-winged macaw described here was diagnosed with CLL based on profound peripheral lymphocytic leukocytosis and supporting results of bone marrow cytology. Treatment with chlorambucil resulted in thrombocytopenia, which resolved with cessation of the drug. These observations suggest that thrombocyte counts should be included in the monitoring of any bird treated with chlorambucil, and thrombocytopenia might be avoided by a treatment interval longer than 3.5 days. The bird was subsequently treated with oral cyclophosphamide and prednisone for 29 weeks and maintained a good quality of life. This case report supports the use of cyclophosphamide and prednisone for CLL in birds, although more long-term studies are needed to better define efficacy.

Acknowledgments. We thank the staff at the Audubon Zoo for their dedicated care of this bird and the staff in the Exotic Animal Ward at the Louisiana State University School of Veterinary Medicine for their assistance with this case. We also thank Dr David Hunley for his assistance with the chemotherapeutic regimen in this case. We also thank the staff at Northwest ZooPath, including Jamie Kinion for data retrieval and Christie Buie for image plating.

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Elizabeth E. Hammond, DVM, David Sanchez-Migallon Guzman, Lic Vet, MS, Dipl ECZM (Avian), Dipl ACZM, Michael M. Garner, DVM, Dipl ACVP, Glenna Mauldin, DVM, MS, Dipl ACVIM, Dipl ACVN, David Martinez-Jimenez, Lic Vet, MSc, Matti Kiupel, DVM, PhD, Dipl ACVP, and Roberto F. Aguilar, DVM

From Lion Country Safari, 2003 Lion Country Safari Rd, Loxahatchee, FL 33470, USA (Hammond); Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Louisiana State University, Skip Bertman Drive, Baton Rouge, LA 70803-8410, USA (Guzman, Mauldin); Northwest ZooPath, 654 W Main, Monroe, WA 98272, USA (Garner); Loving Hands Animal Clinic, 13755 Highway 9, Alpharetta, GA 30004, USA (Martinez-Jimenez); Diagnostic Center for Population and Animal Health, Michigan State University, 4125 Beaumont Rd, Lansing, MI 48910, USA (Kiupel); and Southwest Wildlife Conservation Center, PMB 115 8711 E Pinnacle Peak Rd, Scottsdale, AZ 85255, USA (Aguilar). Present address: Western Veterinary Specialist Centre, 1802 10th Ave SW, Calgary, Alberta, T3C 0J8 Canada (Mauldin); Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California Davis, One Shields Ave, Davis, CA 95616, USA (Guzman).
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