T-cell chronic lymphocytic leukemia in a double yellow-headed Amazon parrot (Amazona ochrocephala oratrix).

Abstract: An adult, male double yellow-headed Amazon parrot (Amazona ochrocephala oratrix) was diagnosed with chronic lymphocytic leukemia based on results of a complete blood cell count and cytologic examination of a bone marrow aspirate. Treatment with oral chlorambucil was attempted, but no response was evident after 40 days. The bird was euthanatized, and the diagnosis of chronic lymphocytic leukemia was confirmed on gross and microscopic examination of tissues. Neoplastic lymphocytes were found in the bone marrow, liver, kidney, testes, and blood vessels. Based on CD3-positive immunocytochemical and immunohistochemical immunophenotyping, the chronic lymphocytic leukemia was determined to be of T-cell origin.

Key words: chronic lymphocytic leukemia, T-cell, chemotherapy, chlorambucil, avian, Amazon parrot, Amazona ochrocephala oratrix

Clinical Report

An adult, male double yellow-headed Amazon parrot (Amazona ochrocephala oratrix), weighing 472 g and at least 13 years old, was referred to the William R Pritchard Veterinary Medical Teaching Hospital at the University of California, Davis, CA, USA, for further diagnostic testing and possible treatment of chronic lymphocytic leukemia (CLL) diagnosed by the referring veterinarian.

The owners did not know whether the bird was wild caught or domestically bred. The bird was the only bird in the household and had no exposure to other birds. It had a 3-week history of decreased appetite and activity level and progressive right pelvic limb lameness. It had previously been an apparently healthy bird aside from a historic fracture of the left tibiotarsal bone and partial digit amputation of digits 2 and 4 of the left foot.

On physical examination, the bird was depressed; mildly dehydrated, based on mildly dry oral and cloacal mucous membranes; and slightly thin, based on the pectoral musculature. Mild episcleral congestion was visible bilaterally, and the oral and cloacal mucous membranes were tinged purple. The bird had decreased weight bearing on the right pelvic limb and was ambulating on the medial aspect of the foot; the bird could grip with the foot, but grip strength was reduced. The remainder of the examination was unremarkable.

Initial diagnostic testing included a complete blood cell count (CBC), plasma biochemical profile, serum bile acid concentration, plasma protein electrophoresis, fecal Gram's stain, whole-body radiographs, and bone marrow aspirate and cytology. Whole-body radiographs and the bone marrow aspirate were performed under general anesthesia with isoflurane and oxygen.

Results of the CBC revealed a normal hematocrit (41%; reference range, 40%-53%) and a marked leukocytosis (white blood cells 391 000/ [micro]L; reference range, 120(-3960/[micro]L), due almost exclusively to a mature lymphocytosis (Table 1). (1-3) The lymphocyte nuclei were small and mature, with clumped chromatin and inapparent nucleoli (Fig 1). The lymphocytes had a small volume of pale cytoplasm, which frequently formed small blebs or pseudopods. There were low numbers of slightly larger lymphocytes that appeared more immature than the others, and immature, nucleated, erythroid precursors were also observed in the

smear. The CBC findings were considered indicative of CLL. Results of the biochemical profile and protein electrophoresis were relatively unremarkable; some values were outside published reference intervals, but they were considered clinically insignificant or within the reference intervals for the university laboratory (Table 1). The bile acid concentration was elevated at 312 [micro]mol/L (reference interval, 35-144 [micro]mol/L). (3)

Results of the fecal Gram's stain revealed normal flora. On survey radiographs, abnormalities were a healed, malunion fracture of the left tibiotarsal bone, chronic left stifle and coxofemoral luxation, bony remodeling of the left coracoid bone consistent with previous trauma, medullary hyperostosis of the right tibiotarsal bone, and a rounded liver. The findings of a rounded liver radiographically and the increased bile acid concentration were suggestive of hepatopathy.

A bone marrow aspirate, taken from the right proximal tibiotarsus, was cellular with numerous unit particles. Most of the cells present were well-differentiated lymphocytes (Fig 2C); scattered erythroid precursors that had complete and synchronous maturation were also present. Similarly, the low numbers of myeloid lineage cells showed complete and orderly maturation, including scattered mature heterophils and eosinophils. The bone marrow results were diagnostic for small, mature lymphocytic neoplasia and considered most consistent with CLL.

Immunocytochemistry was performed on the peripheral blood smear and the bone marrow aspirate with antibodies versus CD3 (CD-3 epsilon, clone CD-3-12, Serotec, Division of MorphoSys, Planegg, Germany) and CD79a (HM57, Dako, Carpinteria, CA, USA) and an appropriate negative control. Results of the CD79a and the negative control immunostains were negative. The small, mature, round cells strongly expressed CD3. As expected, granulocytes and erythroid precursors were negative with the anti-CD3 antibody. The immunocytochemistry results indicated T-cell CLL.

Treatment was initiated using chlorambucil (compounded by Vetricare Rx, Templeton, CA, USA) at a dose of 2 mg/kg PO via gavage feeding needle. The dose of chlorambucil was chosen based on the doses used in other case reports in which lymphoid neoplasms in pet birds were treated. (4,5) Treatments were administered twice weekly, and CBCs were evaluated once weekly (Table 1). Biochemical profiles and bile acid concentrations were evaluated approximately every other week during treatment to monitor for adverse effects secondary to chemotherapy or evidence of disease progression. The bird was also treated with amoxicillin and clavulanic acid (125 mg/kg PO q12h) in case of myelosuppression induced by disease or chemotherapy.

The bird was treated for 40 days; during that time, the lymphocyte counts ranged from 327,320/ [micro]L to 475,200/[micro]L (Table 1). The lymphocyte morphology on repeat CBCs was similar to that on the first CBC until day 40, when the numbers of lymphocytes present that appeared more immature increased slightly. The heterophil counts varied from 0/[micro]L to 4800/[micro]L (reference range, 3300-8800/ [micro][L.sup.3]). Although, on results from one CBC panel, the heterophil count was 0, subjectively, the number of heterophils on each of the peripheral blood smears was similar. The variability was likely because the differential counts were determined by counting 100 leukocytes, and with such massive lymphocytosis, it is possible to have no heterophils in a count of 100 cells, even though the true heterophil count is within the reference interval. Absolute thrombocyte counts were not provided by the laboratory, but subjectively, the thrombocytes appeared decreased on several of the peripheral blood smears starting on day 19. By day 40, the thrombocytes appeared markedly decreased. The hematocrit ranged from 37%-44% during the course of treatment, with mild anemia found on several of the CBC panel results. The serum bile acid concentrations remained increased at 267 [micro]mol/L when rechecked on day 33 of treatment. The plasma aspartate aminotransferase was also high at 570 IU/L (reference range, 120-320 IU/[L.sub.3]) at that time. During the treatment period, no improvement in clinical signs was noted by the owners or the clinicians treating the bird. In addition, the CBC results did not show a response to treatment. As the bird was not improving clinically or hematologically, chemotherapy that was more aggressive was recommended but was declined by the owners.

Fifty-five days after starting treatment, the owners elected euthanasia because the bird's condition appeared to be worsening. A complete necropsy was performed. On gross examination, diffuse hepatomegaly was evident. Multiple, irregularly shaped, mottled, white-tan areas were present throughout the hepatic parenchyma. Similar pale foci were also evident in the kidneys and testes. Tissues were fixed in 10% neutral-buffered formalin, embedded in paraffin, sectioned at 5 [micro]m, and stained with hematoxylin and eosin. Microscopically, white foci in the liver comprised dense infiltrates of pleomorphic neoplastic round cells, which extended from the portal regions into the hepatic parenchyma dissecting along the hepatic sinusoids. Neoplastic cells formed sheets replacing, and occasionally entrapping, degenerate hepatocytes. The neoplastic cells were small and round; had scant, pale eosinophilic cytoplasm; and contained round to oval, hyperchromatic nuclei. Nuclear:cytoplasm ratio was high, and rare mitotic figures were scattered throughout the dense sheets of cells. Similar infiltrates of small, neoplastic, round cells were seen in the testes and kidneys and replaced much of the hematopoietic tissue within the marrow of the long bones (Fig 2A and B). Small to medium blood vessels in the brain, lungs, heart, and skeletal muscles were completely filled with neoplastic cells. Immunohistochemistry was performed on liver and bone marrow sections. The neoplastic cells stained positive with antibodies to CD3 and did not stain with antibodies versus CD79a. These findings were consistent with the previous diagnosis of T-cell CLL.

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[FIGURE 2 OMITTED]

Discussion

In this report, we describe a case of CLL with a T-cell origin in an Amazon parrot. In captive birds, lymphoid neoplasia is the most common form of hemolymphatic neoplasia diagnosed. (6,7) Of the lymphoid neoplasms, the most common in psittacine and passerine birds is multicentric lymphosarcoma. (8) In contrast to lymphosarcoma, lymphocytic leukemia appears to be rare in birds. (6,9)

Lymphosarcoma and lymphocytic leukemia are traditionally differentiated, in part, by tissue of origin. Lymphosarcoma generally arises in secondary lymphoid tissue, whereas leukemia most often originates in the bone marrow. (7) Chronic lymphocytic leukemia is a neoplastic clonal proliferation of small and mature-appearing lymphocytes that manifests as a persistent, often marked, peripheral lymphocytosis. (10) However, in people, CLL and small lymphocytic lymphoma are considered simply to be different manifestations of the same disease, with predominant blood involvement differentiating CLL from small lymphocytic lymphoma. (11,12) In addition, CLL in dogs can arise from the spleen as well as the bone marrow. (13) As a result, there is no clear consensus on making a diagnosis of CLL in veterinary medicine. (14) Chronic lymphocytic leukemia can be of B-cell (B lymphocytes) or T-cell (T lymphocytes) origin; in dogs, T-cell CLL is most common, (l0) In people, B-cell CLL is most common, and the rare entity of a neoplastic clonal proliferation of small and mature-appearing T-cell lymphocytes that used to be called T-cell CLL has been renamed T-prolymphocytic leukemia. (11)

To our knowledge, this is the first reported case of CLL with a T-cell origin in an Amazon parrot and the second reported case of T-cell originated CLL in a psittacine bird. (15) There are several case reports in which immunophenotyping was used to diagnose T-cell lymphoid neoplasia in a variety of nonpoultry avian species. (15-19) Likewise, there have been several case reports in which leukemia has been present associated with a lymphoid neoplasm. (9,15,20-24) Of these reports, only one bird was diagnosed with T-cell CLL, a 32-year-old male green-winged macaw (Ara chloroptera). In the 3 other case reports in which leukemia was identified in the absence of solid lymphoid neoplasia elsewhere in the body, all birds had severe leukocytoses (291 600/[micro]L to approx 2 000 000/[micro]L), consisting primarily of lymphocytes. (9,20,24) Unfortunately, the lymphocyte morphology is only described in one of those cases, a case of a lymphoproliferative disorder in a great-horned owl (Bubo virginianus). In the owl, numerous lymphoblasts with large, eccentric, round nuclei; coarsely granular chromatin; prominent nucleoli; and scant cytoplasm were present. Bone marrow evaluation was nondiagnostic, and the authors were unable to determine the source of the lymphocyte proliferation. (20) Although those 3 cases are consistent with possible primary leukemia, the lymphocytes were not described or were more consistent with acute lymphoblastic leukemia than they were with CLL. Thus, none of the reports, other than that of the green-winged macaw, identified a T-cell lymphoid neoplasm with characteristics consistent with a diagnosis of CLL.

Before arriving at a diagnosis of CLL, the clinician must determine whether the patient has a neoplastic or inflammatory cause for lymphocytosis, especially if the hematologic abnormalities or clinical presentation do not make CLL an obvious conclusion; immunophenotyping by flow cytometry can help with this differentiation as neoplastic lymphocytoses show minimal phenotypic diversity. (14) Molecular genetic analysis of antigen receptor gene rearrangement can be used to assess clonality and to confirm a diagnosis of lymphoid neoplasia; that analysis requires species-specific antigen receptor DNA sequences, which are not readily available for most avian species. (10,13) Once a malignancy has been diagnosed, it must be determined whether the disease is primary leukemia or lymphoma. (14) As mentioned above, the criteria for classifying an individual animal's mature lymphocytic neoplasm as CLL versus leukemic small cell lymphoma are poorly defined. (14) One review states that, in CLL, bone marrow should consist of at least 30% small and mature-appearing lymphocytes. (10) In people, the distinction is based on primary tissue involvement, with primary blood involvement indicating CLL. (11) In the case we describe, the white blood cell count was profoundly elevated and consisted primarily of small, mature lymphocytes, making the diagnosis of malignancy straightforward. The bone marrow aspirate sample consisted primarily of small, mature lymphocytes, consistent with a diagnosis of CLL.

Chronic lymphocytic leukemia can be further classified as having a T-cell or B-cell origin by immunophenotyping. Most immunophenotyping is performed with monoclonal antibodies. (10) The most common monoclonal antibodies used in veterinary medicine to immunophenotype lymphocytes are antibodies versus CD3, which is expressed by T cells, and versus CD79a, which is expressed throughout all stages of B-cell development. (10) CD3 is highly conserved across many species and anti-CD3 antibodies have been shown to accurately stain T cells in chickens. (25) In contrast, the anti-B-cell antibodies tested (B29, anti mb-1) were not able to accurately identify B cells in chickens. (25) Immunophenotyping has been used in many of the case reports on nondomestic avian species with lymphoma but has generally not been validated for use in those species. (16-19,21,26) For example, B-lymphocyte antigen antibodies (BLA.36) were used to diagnose B-cell neoplasia in 2 umbrella cockatoos (Cacatua alba). (21,26) In another case report of T-cell intestinal lymphosarcoma in a yellow-naped Amazon parrot (Amazona ochrocephala auropalliata), CD79a and BLA.36 expression were not seen in control psittacine-bird splenic tissue or neoplastic tissue, whereas CD3 staining was found in both. (17) In the case reported here, we are fairly confident of the positive immunophenotyping with CD3, allowing the diagnosis of T-cell CLL. However, in our experience, none of the reagents tested at the University of California, Veterinary Medical Teaching Hospital have thus far been able to reliably identify avian B cells, including antibodies versus CD79a and BLA.36.

Clinical features associated with CLL in people can include lymphadenopathy, hepatosplenomegaly, anemia, and thrombocytopenia. (27) The anemia and thrombocytopenia can be caused by myelophthisis; however, in people with CLL, immune-mediated cytopenias are also common. (11,27) In dogs and cats, CLL is most commonly diagnosed as an incidental finding when blood tests are performed for other reasons. (10) In dogs that are clinically ill with CLL, the most common clinical signs are lethargy, reduced appetite, weight loss, hepatosplenomegaly, and pale mucous membranes if moderate to severe anemia is present. (10) Mild thrombocytopenia is also a common finding in dogs and cats with CLL. (10) In birds with lymphoid neoplasia, varied presentations and clinical findings have been described. (7) In the case reports of nonpoultry birds in which a severe lymphoid leukemia was diagnosed, the clinical findings consisted of lethargy, reduced ability to fly, and reduced appetite or body condition; one bird had clinical signs associated with the respiratory tract but concurrent pneumonia and heart failure were present. (9,20,24) In the case of the green-winged macaw, CLL was initially diagnosed from results of routine blood tests during an annual examination. (15) In the case we describe, the parrot was presented for reduced appetite and energy level and progressive right pelvic limb lameness; the bird was mildly thin on physical examination. Aside from the right pelvic limb lameness, this presentation is consistent with CLL across species.

Anemia and thrombocytopenia have been reported to be common findings in birds with lymphoid leukemia and anemia was present in most of the case reports of lymphoid neoplasia in nonpoultry species. (7,9,l5,16,18,20,21,28) The bird we describe had an intermittent mild anemia; however, thrombocytopenia was more persistent and progressive, although thrombocytes were judged to be adequate before starting therapy. Likely causes of the anemia included myelophthisis, bone marrow suppression secondary to chemotherapy, and anemia of chronic disease (29) The decreased thrombocytes were presumably caused by either myelophthisis secondary to the CLL or bone marrow suppression secondary to chlorambucil. (10,11,30) The green-winged macaw with CLL was suspected of experiencing thrombocytopenia secondary to chlorambucil administration. (15) As stated above, immune-mediated cytopenias are common in people with CLL, but, to our knowledge, they have never been reported in birds.

Hyperglobulinemia is another potential abnormality in patients with CLL. In humans with B-cell CLL, monoclonal hypergammaglobulinemia can occur. (31,32) In humans with angioblastic T-cell lymphoma, a peripheral T-cell lymphoma, polyclonal hypergammaglobulinemia is invariably present. (33) In one study, a high percentage of dogs with CLL were reported to have a monoclonal gammopathy, but immunophenotyping was not performed. (34) This finding is unexpected because monoclonal gammopathy is more likely to be present in B-cell CLL, but T-cell CLL is more common than B-cell CLL in dogs. (10) Other studies in dogs with CLL have found a low percentage of monoclonal gammopathies. (10) The Amazon parrot in this report had normal results on plasma protein electrophoresis, which is not surprising given the diagnosis of T-cell CLL. The green-winged macaw diagnosed with T-cell CLL had an increased [beta]-globulin concentration and a marked hypergammaglobulinemia on plasma protein electrophoresis; however, the authors did not indicate whether the hypergammaglobulinemia was polyclonal or monoclonal. (15) That bird was also suspected of having aspergillosis, which could have caused the changes in the globulin levels. (35) More cases of CLL in birds will need to be evaluated to understand the frequency and significance of globulin changes in this disease.

Hepatomegaly, splenomegaly, or both have been a common finding in nondomestic birds diagnosed with lymphoid neoplasia. (15,16,18,19,36-45) The kidneys are the next most commonly involved organ.(7,15,28) In the present case, the parrot had neoplastic lymphocytes in the liver, kidneys, testes, bone marrow, and peripheral blood. Aside from the testes, those findings are consistent with lymphoid neoplasia in nonpoultry species. The elevations in bile acid concentration and the rounded liver on radiographs are both consistent with neoplastic involvement of the liver found at necropsy.

The causes of the right pelvic limb lameness, episcleral congestion, and purple-tinged mucous membranes in the Amazon parrot remain unclear. Those are not common findings in birds with lymphoid neoplasia or in mammals with CLL. However, at necropsy, the small- to medium-sized vessels in many of the organs of this bird were completely filled with neoplastic cells. The profound lymphocytosis possibly was adversely affecting blood flow and oxygenation of the tissues, leading to changes in appearance of vessels and mucous membranes. The right pelvic limb lameness could have been caused by bone pain secondary to myelophthisis, although other causes, such as trauma resulting from generalized weakness, cannot be ruled out. Radiographs revealed hyperostosis of the right tibiotarsus; hyperostosis can occur secondary to reproductive hormone production associated with egg laying, ovarian cysts, and possibly, reproductive neoplasms, metastatic neoplasia, certain infections (eg, mycobacteriosis), osteopetrosis, hypertrophic osteopathy, and metabolic bone disease. (46,47) Interestingly, osteopetrosis is caused by the avian leukosis/sarcoma group of viruses in poultry and is frequently associated with lymphoid leukosis (a lymphoid neoplasia with a leukemic component). There were no necropsy findings that would explain the right pelvic limb lameness, other than the CLL expansion within the marrow cavities of the long bones. (48)

In humans, CLL has historically been treated with the alkylating agent chlorambucil, and that drug is often still used in elderly patients. (11,27,30) Alkylating agents are not cell cycle-specific and are cross-linked to cellular DNA, which can cause cell death. (30,49) The primary toxicity of chlorambucil is myelosuppression. (30) Newer chemotherapeutic combinations are generally used now, such as rituximab, an anti-CD20 monoclonal antibody, combined with fludarabine, a purine analog, and cyclophosphamide, an alkylating agent, (11,27,30) More aggressive and sophisticated therapies, such as stem cell transplantation, are also being used with increasing frequency. (27) Treatment choice depends on the staging of the disease, the molecular profile of the individual tumor, patient age, and the performance status of the patient. (11,27)

In dogs, treatment may not be pursued because the disease can be fairly indolent. However, if treatment is indicated, chlorambucil, with or without prednisone, is generally the initial therapy. In patients that do not respond to chlorambucil, additional chemotherapeutic drugs, such as vincristine or cyclophosphamide, or multidrug protocols as used in high-grade lymphoma, have been used. (10)

In the few cases of nonpoultry birds with primary leukemia, varying treatment protocols have been attempted. Avian oncology is still in its infancy, so treatment protocols often have to be extrapolated from other species. (50) Combination chemotherapy consisting of vincristine sulfate, prednisone, and chlorambucil did not lead to remission in a pekin duck (Anas platyrhynchos) with lymphocytic leukemia. (9) An apparent reported success in treating leukemia in a bird was complete remission of lymphoid leukemia in a saker falcon (Falco cherrug) treated with potassium arsenite for 10 days; however, that patient was only followed for 1 month before publication of the case report. (24) The green-winged macaw with T-cell CLL was treated initially with chlorambucil and prednisone until thrombocytopenia occurred; the chemotherapy was then changed to cyclophosphamide and prednisone. The bird's severe leukocytosis and apparent good quality of life remained stable for 29 weeks while on the cyclophosphamide-prednisone combination, but the bird deteriorated fairly rapidly once chemotherapy was stopped because of hurricane Katrina. (15)

In this case, single-drug therapy with chlorambucil was chosen because chlorambucil is commonly used in treating both humans and dogs with CLL. In addition, it has manageable toxicity, is easy to administer, and was not considered likely to affect the bird's quality of life, which was important to the bird's owners. Steroids were not used at the start of treatment because of the increased risk of steroid use in birds and because corticosteroids do not lead to actual increases in remission duration in people. (30,51) The chlorambucil dosing regimen was based on 2 other case reports that used similar dosing with minimal side effects. (4,5,21) However, the chlorambucil treatment regimen did not lead to any changes in the bird's clinical signs or improvement in the severe leukemia. The disease likely was quite advanced at the time of diagnosis, given the magnitude of the lymphocytosis, the presence of clinical signs, and the infiltration of the liver, kidneys, and testes at the time of necropsy. In humans and dogs with B-cell CLL, total lymphocyte count does not appear to impact survival time. (14) In contrast, one study found that dogs with T-cell CLL with an initial lymphocyte count greater than 30 000/[micro]L had a much shorter median survival time than those with lymphocytes of fewer than 30 000/[micro]L. (14) The bird in this case report had an initial lymphocyte count of 387 090/[micro]L, but the prognostic significance of this in birds is unknown at this time. Combination chemotherapy could have led to a longer survival time in this case and was recommended when no response to the chlorambucil was evident; however, the owners declined. It is not clear whether the bird suffered any toxic side effects of the chlorambucil. The thrombocytes appeared markedly decreased by the time of euthanasia and mild anemia was present on several of the CBCs. The mild anemia and the thrombocytopenia could have been caused by myelophthisis or chlorambucil toxicity, although heteropenia would likely also be expected in these instances. The green-winged macaw treated with chlorambucil for CLL did develop thrombocytopenia that resolved with cessation of chlorambucil therapy. (15) In addition, the bird we describe had some changes in liver analytes (aspartate aminotransferase, bile acids); chlorambucil therapy has been shown to cause hepatopathies in people and an elevated alanine aminotransferase concentration in one macaw (Ara ararauna). (4,49) However, the hepatopathy in this Amazon parrot was more likely caused by neoplastic infiltration of the liver, as there were abnormalities present before initiation of chemotherapy.

Amoxicillin plus clavulanic acid was added to the treatment regimen in this case because of concern for potential disease or chemotherapy induced immunosuppression; secondary infection is common in humans with CLL. (11)

In this Amazon parrot, T-cell CLL was diagnosed likely late in its course, and the relatively conservative chemotherapy that was attempted did not appear to have any effect on the tumor. Either increases in the dose or frequency of administration of chlorambucil or the use of other chemotherapeutic agents may be necessary for successful treatment of this disease in this species. The quality of life of the green-winged macaw diagnosed with CLL in a recent case report was maintained for 29 weeks while on cyclophosphamide and prednisone chemotherapy; however, CLL was diagnosed in this bird before onset of clinical signs, so it is unclear how the disease would have progressed without treatment. (15) Regardless, further therapeutic trials will be needed to determine the optimal treatment regimen for T-cell CLL in avian species.

References

(1.) Carpenter J. Exotic Animal Formulary. 3rd ed. St Louis, MO: Elsevier; 2005.

(2.) Airman RB, Clubb SL, Dorrestein GM, Quesenberry K, eds. Avian Medicine and Surgery. Philadelphia, PA: WB Saunders; 1997.

(3.) Fudge AM. Laboratory Medicine Avian and Exotic Pets. Philadelphia, PA: WB Saunders Co; 2000.

(4.) Kollias GV, Homer B, Thompson JP. Cutaneous pseudolymphoma in a juvenile blue and gold macaw (Ara ararauna). J Zoo Wildl Med. 1992; 23(2):235-240.

(5.) Rivera S, Reavill DR, McClearen JR. Treatment of cutaneous lymphosarcoma in an umbrella cockatoo (Cacatua alba). Proc Annu Conf Assoc Avian Vet. 2002:99-100.

(6.) Latimer KS. Oncology. In: Ritchie BW, Harrison GJ, Harrison L, eds. Avian Medicine." Principles and Application. Lake Worth, FL: Wingers Publishing; 1994:640-672.

(7.) Coleman CW. Lymphoid neoplasia in pet birds: a review. J Avian Med Surg. 1995;9(1):3-7.

(8.) Reavill DR. Tumors of pet birds. Vet Clin North Am Exot Anim Pract. 2004;7(3):537-560.

(9.) Newell SM, McMillan MC, Moore FM. Diagnosis and treatment of lymphocytic leukemia and malignant lymphoma in a Pekin duck (Anas platyrhynchos domesticus). J Assoc Avian Vet. 1991;5(2):83-86.

(10.) Workman HC, Vernau W. Chronic lymphocytic leukemia in dogs and cats: the veterinary perspective. Vet Clin North Am Small Anim Pract. 2003; 33(6):1379-1399.

(11.) Gribben JG. How I treat CLL up front. Blood. 2010;115(2): 187-197.

(12.) Harris NL, Jaffe ES, Diebold J, et al. World Health Organization classification of neoplastic diseases of the hematopoietic and lymphoid tissues: report of the Clinical Advisory Committee meeting--Airlie House, Virginia, November 1997. J Clin Oncol. 1999;17(12):3835-3849.

(13.) Vernau W, Moore PF. An immunophenotypic study of canine leukemias and preliminary assessment of clonality by polymerase chain reaction. Vet Immunol Immunopathol. 1999;69(24): 145-164.

(14.) Avery AC, Avery PR. Determining the significance of persistent lymphocytosis. Vet Clin North Am Small Anim Pract. 2007;37(2):267-282.

(15.) Hammond EE, Sanchez-Migallon Guzman D, Garner MM, et al. Long-term treatment of chronic lymphocytic leukemia in a green-winged macaw (Ara chloroptera). J Avian Med Surg. 2010;24(4): 330-338.

(16.) Burgos-Rodriguez AG, Garner MM, Ritzman TK, Orcutt CJ. Cutaneous lymphosarcoma in a double yellowheaded Amazon parrot (Amazona ochrocephala oratrix). J Avian Med Surg. 2007;21(4): 283-289.

(17.) Souza M J, Newman S J, Greenacre CB, et al. Diffuse intestinal T-cell lymphosarcoma in a yellow-naped Amazon parrot (Amazona ochrocephela auropalliata). J Vet Diagn Invest. 2008;20(5): 656-660.

(18.) de Wit M, Schoemaker N J, Kik M J, Westerhof I. Hypercalcemia in two Amazon parrots with malignant lymphoma. Avian Dis. 2003;47(1): 223-28.

(19.) Malka S, Crabbs T, Mitchell EB, et al. Disseminated lymphoma of presumptive T-cell origin in a great horned owl (Bubo virginianus). J Avian Med Surg. 2008;22(3):226-233.

(20.) Kelly TR, Vennen KM, Duncan R, Sleeman JM. Lymphoproliferative disorder in a great horned owl (Bubo virginianus). J Avian Med Surg. 2004; 18(4):263-268.

(21.) Rivera S, McClearen JR, Reavill DR. Treatment of nonepitheliotropic cutaneous B-cell lymphoma in an umbrella cockatoo (Cacatua alba). J Avian Med Surg. 2009;23(4):294-302.

(22.) Campbell T. Lymphoid leukosis in an Amazon parrot--a case report. Intl Conf Avian Med. 1984: 229-234.

(23.) France M, Gilson S. Chemotherapy treatment of lymphosarcoma in a Moluccan cockatoo. Proc Annu Conf Assoc Avian Vet. 1993:15-19.

(24.) Tarello W. Lymphoid leukaemia in a saker falcon. Vet Rec. 2006; 158(6):212.

(25.) Jones M, Cordell JL, Beyers AD, et al. Detection of T and B cells in many animal species using crossreactive anti-peptide antibodies. J Immunol. 1993;150(12):5429-5435.

(26.) Latimer KS, Ritchie BW, Campagnoli RP, Harris DJ. Cutaneous T-cell-rich B-cell lymphoma and leukemic blood profile in an umbrella cockatoo (Cacatua alba). Paper presented at: Annual International Virtual Conference in Veterinary Medicine; 1998; http://www.vet.uga.edu/ivcvm/. Accessed March 26, 2010.

(27.) Herishanu Y, Polliack A. Chronic lymphocytic leukemia: a review of some new aspects of the biology, factors influencing prognosis and therapeutic options. Transfus Apher Sci. 2005;32(1): 85-97.

(28.) Reavill D. Pet bird oncology. Proc Annu Conj Assoc Avian Vet. 2001:29-43.

(29.) Fudge AM. Disorders of avian erythrocytes. In: Fudge AM, ed. Laboratory Medicine Avian and Exotic Pets. Philadelphia, PA: WB Saunders; 2000: 28-34.

(30.) Wilhelm K, Yang D. A review of pharmacologic options for previously untreated chronic lymphocytic leukemia. J Oncol Pharm Pract. 2011;17(2): 91-103.

(31.) Keating MJ. Chronic lymphocytic leukemia. Semin Oncol. 1999;26(5, suppl 14):107-114.

(32.) Guastafierro S, Celentano M, Cuomo C, Falcone U. Chronic lymphocytic leukemia with associated lambda-light-chain and IgG lambda paraproteins simulating a biclonal gammopathy. Clin Lab. 2010; 56(11 12):577-580.

(33.) Bal M, Gujral S, Gandhi J, et al. Angioimmunoblastic T-cell lymphoma: a critical analysis of clinical, morphologic and immunophenotypic features. Indian J Pathol Microbiol. 2010;53(4):640-645.

(34.) Leifer CE, Matus RE. Chronic lymphocytic leukemia in the dog: 22 cases (1974-1984). J Am Vet Med Assoc. 1986;189(2):214-217.

(35.) Rosenthal KL. Avian protein disorders. In: Fudge AM, ed. Laboratory Medicine Avian and Exotic Pets. Philadelphia, PA: WB Saunders; 2000: 171-173.

(36.) Turrel M J, McMillan MC, Paul-Murphy J. Diagnosis and treatment of tumors of companion birds II. AAV Today. 1987;1(4):159-165.

(37.) Paul-Murphy J, Lowenstine L, Turrel JM, et al. Malignant lymphoreticular neoplasm in an African gray parrot. J Am Vet Med Assoc. 1985;187(11): 1216-1217.

(38.) Bauck L. A clinical approach to neoplastic disease in the pet bird. Semin Avian Exot Pet Med. 1992; 1(2):65-72.

(39.) Wadsworth PF, Jones DM, Pugsley SL. Some cases of lymphoid leukosis in captive wild birds. Avian Pathol. 1981 ; 10(4):499-504.

(40.) Rambow VJ, Murphy CJ, Fox JG. Malignant lymphoma in a pigeon. J AmVet Med Assoc. 1981; 179(11):1266-1268.

(41.) Wade LL, Polack EW, O'Connell PH, et al. Multicentric lymphoma in a European starling (Sturnus vulgaris). J Avian Med Surg. 1999;13(2): 108-115.

(42.) Coleman C, Oliver R. Lymphosarcoma in a juvenile blue and gold macaw (Ara ararauna) and a mature canary (Serinus canarius). J Assoc Avian Vet. 1994;8(2):64-68.

(43.) Palmer GH, Stauber E. Visceral lymphoblastic leukosis in an African gray parrot. Vet Med: Small Anita Clin. 1981;76(9):1355-1356.

(44.) Bauck L. Lymphosarcoma/avian leukosis in pet birds-case reports. Proc Annu Conf Assoc Avian Vet. 1986:241-45.

(45.) Graham DL. Lymphoreticular and hematopoietic proliferative lesions in non-domestic avian species a survey of diagnostic submissions. Proc Annu Conf Assoc Avian Vet. 1995:461-464.

(46.) Baumgartner R, Hatt J, Dobeli M, Hauser B. Endocrinologic and pathologic findings in birds with polyostotic hyperostosis. J Avian Med Surg. 1995;9(4):251-254.

(47.) Quesenberry K, Orosz S, Dorrestein GM. Musculoskeletal system. In: Altman RB, Clubb SL, Dorrestein GM, Quesenberry K, eds. Avian Medicine and Surgery. Philadelphia, PA: WB Saunders; 1997:517-539.

(48.) Calnek B. Neoplastic diseases. In: Calnek B, ed. Diseases of Poultry. 10th ed. Ames: Iowa State University Press; 1997:367-413.

(49.) Plumb D. Plumb's Veterinary Drug Handbook. 6th ed. Ames, IA: Blackwell Publishing; 2008.

(50.) Filippich LJ. Tumor control in birds. Semin Avian Exot Pet Med. 2004; 13(1):25-43.

(51.) Quesenberry KE, Hillyer EV. Supportive care and emergency therapy. In: Ritchie BW, Harrison GJ, Harrison LR, eds. Avian Medicine." Principles and Application. Lake Worth, FL: Wingers Publishing; 1994:382-416.

Anna Osofsky, DVM, Dipl ABVP (Avian), Michelle G. Hawkins, VMD, Dipl ABVP (Avian), Oded Foreman, DVM, Dipl ACVP, Michael S. Kent, MAS, DVM, Dipl ACVIM (Oncology), Dipl ACVR (Radiation Oncology), William Vernau, BSc, BVMS, DVSc, PhD, Dipl ACVP, and Linda J. Lowenstine, DVM, PhD, Dipl ACVP

From the William R Pritchard Veterinary Medical Teaching Hospital, One Shields Ave, Davis, CA 95616, USA (Osofsky, Foreman); and the Departments of Medicine and Epidemiology (Hawkins), Surgical and Radiological Sciences (Kent), and Pathology, Microbiology, and Immunology (Vernau, Lowenstine), School of Veterinary Medicine, One Shields Ave, University of California, Davis, CA 95616, USA. Present address: Carrollton West Pet Hospital, 3729 Old Denton Rd, Carrollton, TX 75007, USA (Osofsky); Jackson Laboratory, 4910 Raley Blvd, Sacramento, CA 95838, USA (Foreman).

Table 1. Results of the complete blood cell count, plasma biochemical
profile, and plasma protein electrophoresis in an Amazon parrot
diagnosed with chronic lymphocytic leukemia.

                                            Day of treatment

Test Parameter                       1            12            19

Complete blood cell count

Hematocrit (%)                        41            44             38
MCV (fL)                             163.3         156.6          154.5
MCH (pg)                              43.4          43.1           42.7
MCHC (g/dL)                           26.6          27.5           27.6
Anisocytosis                    Moderate        Slight         Slight
Polychromasia                   Moderate        Slight         Slight
White blood cells                 391000        376000         340000
  (cells/[micro]L)
Bands (cells/[micro]L)                 0             0              0
Heterophils (cells/[micro]L)        3910          3760           3400
Lymphocytes                       387090        372240         336600
  (cells/[micro]L)
Monocytes (cells/[micro]L)             0             0              0
Eosinophils (cells/[micro]L)           0             0              0
Basophils (cells/[micro]L)             0             0              0
Thrombocytes                    Adequate      Adequate      Decreased

Plasma fibrinogen                    100           400            400
  (mg/dL)

Plasma biochemical analysis

Albumin (g/dL)                         1            --              1
AST (IU/L)                           369            --            304
Bile acids ([micro]mol/L)            312            --          --
Calcium (mg/dL)                        8.9          --              9.6
Cholesterol (mg/dL)                  272            --            203
Creatinine kinase                    171            --            205
  (IU/L)
Globulin (g/dL)                        2.7          --              3.0
Glucose (mg/dL)                      406            --            457
LDH (IU/L)                           148            --            141
Phosphorus (mg/dL)                     2.7          --              3.3
Total protein (g/dL)                   3.7          --              4.0
Uric acid (mg/dL)                      3.3          --              2.8

Plasma protein electrophoresis

A/G ratio                              1.63         --          --
Pre-albumin (g/dL)                     0.58         --          --
Albumin (g/dL)                         2.02         --          --
Alpha 1 globulin                       0.29         --          --
  (g/dL)
Alpha 2 globulin                       0.29         --          --
  (g/dL)
Beta globulin (g/dL)                   0.6          --          --
Gamma globulin                         0.42         --          --
  (g/dL)

                                           Day of treatment

Test Parameter                      26            30            33

Complete blood cell count

Hematocrit (%)                         38           38             37
MCV (fL)                              149.6        154.5          151.6
MCH (pg)                               42.5         42.3           42.2
MCHC (g/dL)                            28.4         27.4           27.8
Anisocytosis                       Slight       Slight       Moderate
Polychromasia                    Moderate       Slight       Moderate
White blood cells                  380000       400000         334000
  (cells/[micro]L)
Bands (cells/[micro]L)                  0            0              0
Heterophils (cells/[micro]L)            0         4000           3340
Lymphocytes                        380000       396000         327320
  (cells/[micro]L)
Monocytes (cells/[micro]L)              0            0           3340
Eosinophils (cells/[micro]L)            0            0              0
Basophils (cells/[micro]L)              0            0              0
Thrombocytes                    Decreased     Adequate      Decreased

Plasma fibrinogen                     300          300            200
  (mg/dL)

Plasma biochemical analysis

Albumin (g/dL)                      --              --              1.1
AST (IU/L)                          --              --            570
Bile acids ([micro]mol/L)           --              --            267
Calcium (mg/dL)                     --              --              9.1
Cholesterol (mg/dL)                 --              --            194
Creatinine kinase                   --              --            280
  (IU/L)
Globulin (g/dL)                     --              --              2.8
Glucose (mg/dL)                     --              --            350
LDH (IU/L)                          --              --             74
Phosphorus (mg/dL)                  --              --              2.7
Total protein (g/dL)                --              --              3.9
Uric acid (mg/dL)                   --              --              2.7

Plasma protein electrophoresis

A/G ratio                           --              --      --
Pre-albumin (g/dL)                  --              --      --
Albumin (g/dL)                      --              --      --
Alpha 1 globulin                    --              --      --
  (g/dL)
Alpha 2 globulin                    --              --      --
  (g/dL)
Beta globulin (g/dL)                --              --      --
Gamma globulin                      --              --      --
  (g/dL)

                                  Day of
                                treatment        Reference

Test Parameter                      40        intervals (1-3)

Complete blood cell count

Hematocrit (%)                         43          40-53
MCV (fL)                              174.8       163-170
MCH (pg)                               42.3        28-55
MCHC (g/dL)                            24.2        22-32
Anisocytosis                     Moderate         Slight
Polychromasia                    Moderate         Slight
White blood cells                  480000       6000-11 000
  (cells/[micro]L)
Bands (cells/[micro]L)                  0            0
Heterophils (cells/[micro]L)         4800        3300-8800
Lymphocytes                        475200        1200-3960
  (cells/[micro]L)
Monocytes (cells/[micro]L)              0          0-330
Eosinophils (cells/[micro]L)          230          0-330
Basophils (cells/[micro]L)              0          0-130
Thrombocytes                     Markedly        Adequate
                                decreased
Plasma fibrinogen                     100          <300
  (mg/dL)

Plasma biochemical analysis

Albumin (g/dL)                      --            1.2-2.0
AST (IU/L)                          --            120-320
Bile acids ([micro]mol/L)           --            35-144
Calcium (mg/dL)                     --           8.5-14.0
Cholesterol (mg/dL)                 --            180-305
Creatinine kinase                   --            55-345
  (IU/L)
Globulin (g/dL)                     --            2.3-3.6
Glucose (mg/dL)                     --            250-370
LDH (IU/L)                          --            160-420
Phosphorus (mg/dL)                  --            3.1-5.5
Total protein (g/dL)                --              3-5
Uric acid (mg/dL)                   --            2.3-10

Plasma protein electrophoresis

A/G ratio                           --            1.9-5.9
Pre-albumin (g/dL)                  --           0.35-1.05
Albumin (g/dL)                      --            1.9-3.5
Alpha 1 globulin                    --           0.05-0.32
  (g/dL)
Alpha 2 globulin                    --           0.07-0.32
  (g/dL)
Beta globulin (g/dL)                --           0.12-0.72
Gamma globulin                      --           0.17-0.76
  (g/dL)

Abbreviations: MCV indicates mean corpuscular volume; MCH, mean
corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin
concentration; AST, aspartate aminotransferase; LDH, lactate
dehydrogenase; A/G, albumin/globulin.