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Cutaneous T-cell-rich B-cell Lymphoma and Leukemic Blood Profile in an Umbrella Cockatoo (Cacatua alba)
Kenneth S. Latimer, Branson W. Ritchie, Raymond P. Campagnoli, and Don J. Harris
College of Veterinary Medicine, The University of Georgia, Athens, Georgia 30602 (Latimer, Ritchie, Campagnoli) and South Kendall Animal Hospital, 9501 S.W. 160th Street, Miami, FL 33187 (Harris) USA
Abstract. An adult Umbrella Cockatoo was presented for examination for suspected folliculitis. Skin and feather follicle biopsies contained nodular aggregates of lymphocytes within the dermis and subcutaneous fat. The cell population included large lymphoblasts and small mature lymphocytes. Blood smears had a marked lymphocytosis. Most of the lymphocytes were small and well differentiated, while fewer cells were large lymphocytes and lymphoblasts. Immunohistochemistry demonstrated that the larger neoplastic lymphocytes in skin and blood were B-cells, while the smaller lymphocytes were reactive T-cells. The final diagnosis was cutaneous T-cell-rich B-cell lymphoma with a leukemic blood profile.
Key Words: Avian, Cacatua alba, Umbrella Cockatoo, Cutaneous lymphoma, T-cell-rich B-cell lymphoma, Skin, B-lymphocytes, T-lymphocytes, Immunohistochemistry, Leukemic blood profile, Lymphocytosis
Introduction
Lymphoma is observed frequently in poultry, but is uncommon in companion and free ranging birds. Lymphoid leukosis and Marek’s disease are two forms of lymphoma observed in chickens. Lymphoid leukosis, caused by a retrovirus, is characterized by the presence of lymphoblasts. Neoplasms frequently involve the liver, spleen, and bursa of Fabricius. 1 Marek’s disease, caused by an oncogenic herpesvirus (serotype 1), is associated with widespread neoplasms involving visceral organs and sciatic nerves. 2 Infrequently, Marek’s disease may present as multiple, intact to ulcerated skin nodules involving the feather tracts. 3 Microscopically, the neoplastic cell population may appear inflammatory or may consist of a uniform population of lymphoblasts. Neoplastic cell infiltrates may involve the feather follicle and feather pulp cavity. 2,3
Reports of lymphoma in psittacine birds are infrequent, and most neoplasms have involved visceral organs. 4-14 A leukemic blood profile has been documented in one Amazon Parrot,7 but complete blood cell (CBC) counts have been within reference intervals when performed in three other psittacine birds with lymphoma. 8,9 This case report presents an Umbrella Cockatoo with cutaneous T-cell-rich B-cell lymphoma and a leukemic blood profile.
Case Report
An adult Umbrella Cockatoo (Cacatua alba) of undetermined gender was presented for suspected severe, generalized folliculitis and localized pododermatitis. Palpation of the skin revealed thickened dermal nodules. The gross appearance of the bird’s skin was similar to that described for cutaneous Marek’s disease of chickens. Skin biopsies and blood smears were obtained and submitted to the University of Georgia College of Veterinary Medicine for further evaluation.
Microscopically, sections of skin were covered by an intact stratified squamous epithelium. Scattered feather follicles were present. Both feather follicles and developing feathers were unremarkable. Normal deep dermal architecture was replaced by multifocal to coalescing, perivascular to dermal infiltrates of lymphocytes. Neoplastic infiltrates also extended into the underlying subcutis. Some of the lymphoid infiltrates were arranged in variably-sized nodules (Fig. 1). The lymphoid cell population included large lymphoblasts, numerous small well differentiated lymphocytes, and widely scattered plasma cells (Fig. 2). The mitotic index was moderate. The initial histological diagnosis was cutaneous lymphoma.
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| Fig. 1. Skin, Umbrella Cockatoo, H&E stain. Lymphoid infiltrates in deep dermis and subcutis have a nodular appearance. |
Fig. 2. Skin, Umbrella Cockatoo, H&E stain. Perivascular accumulation of neoplastic lymphocytes include lymphoblasts and small lymphocytes. Scattered mitoses are present. |
Replicate tissue sections were prepared for immunohistochemical staining and DNA in situ hybridization. Formalin-fixed tissue also was prepared for ultrastructural examination. Blood smears were evaluated for the presence or absence of a leukemic blood profile.
Immunohistochemistry was performed by the avidin-biotin complex immunoperoxidase technique using T- and B-cell specific antibodies. a Diaminobenzidine was used as the chromagen. Tissue sections prepared for B-lymphocyte (BLA.36 immunoreactivity) and T-lymphocyte (CD3 immunoreactivity) were not digested prior to staining. However, the tissue sections were pretreated with steam for 30 minutes in the presence of 10mM citrate buffer (pH = 6.0) to improve antigen retrieval. Following immunohistochemical staining, antibody localization was visualized as brown discoloration of the cell membrane (BLA.36) or cytoplasm (CD3). The increased numbers of small, well differentiated cells were reactive T-cells (Fig. 3), while the neoplastic lymphocytes were identified as B-cells. The B-lymphocytes included lymphoblasts, medium- to large-sized lymphocytes, and infrequent plasma cells (Fig. 4). In tissue sections, the T-cells were closely associated, while the B-cells were scattered more diffusely.
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| Fig. 3. Umbrella Cockatoo, skin, CD3 immunoreactivity. Clusters of small T-lymphocytes are present within the neoplasm. |
Fig. 4. Umbrella Cockatoo, skin, BLA.36 immunoreactivity. Lymphoblasts and variably-sized B-cells are scattered more diffusely in the neoplasm. |
Smaller well differentiated lymphocytes within blood vessels were T-cells, while larger lymphocytes and lymphoblasts were identified as B-cells (Figs. 5 & 6).
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| Fig. 5. Umbrella Cockatoo blood, CD3 immunoreactivity. Small lymphocytes exhibit T-cell staining pattern. |
Fig. 6. Umbrella Cockatoo, blood, BLA.36 immunoreactivity. Lymphoblasts and large lymphocytes exhibit B-cell staining pattern. |
DNA in situ hybridization was negative for herpesvirus nucleic acid. Electron microscopic examination of formalin-fixed tissue failed to disclose virions; however, severe fixation artifact was present.
Wright-Leishman-stained blood smears had an estimated total leukocyte count of 38,000 to 42,000 cells /µl. The leukocyte differential count was 94% lymphocytes, 5% segmented heterophils, and 1% monocytes. The absolute lymphocyte count was estimated between 35,720 and 39,480 cells per microliter of blood. Lymphocyte size was quite variable (Figs. 7 & 8).
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| Fig. 7. Umbrella Cockatoo, blood, Wright-Leishman stain. A few lymphoblasts and many well differentiated small lymphocytes are present. |
Fig. 8. Umbrella Cockatoo, blood, Wright-Leishman stain. Large lymphocytes occasionally have prominent blunt pseudopods. |
Approximately 80% of the lymphocytes appeared small and well differentiated , while 20% of the lymphocytes were medium- to large-sized lymphocytes and lymphoblasts. The larger lymphocytes had a finely to moderately dispersed chromatin pattern and a thin rim of dark blue, granular cytoplasm. Some of these cells also had prominent, blunt pseudopods. The lymphoblasts had a similar appearance, but nuclei contained nucleoli or nucleolar rings. Mitotic figures were present but rare. Large aggregates of thrombocytes were present within the smear. Occasional polychromatophilic erythrocytes were observed. The combined hematologic and immunohistochemical observations indicated that lymphocytosis was primarily a reactive response, associated with an abundance of well differentiated T-cells. The leukemic blood profile, however, could be attributed to the presence of lymphoblasts of B-cell lineage. Therefore, the definitive diagnosis in this Umbrella Cockatoo was cutaneous T-cell-rich B-cell lymphoma with a leukemic blood profile.
The bird died approximately six months after diagnosis of cutaneous lymphoma. A necropsy was not allowed.
Discussion
The diagnosis of cutaneous T-cell-rich B-cell lymphoma (TCRBCL) in this cockatoo is supported by histologic observations and immunohistochemical staining patterns. The neoplastic cells lacked epitheliotropism, a characteristic of cutaneous T-cell lymphoma. 15 The immunoreactivity of the tissue sections indicated that T-lymphocytes were more numerous and well differentiated, while the B-lymphocytes often were blasts or large lymphocytes that were more widely scattered in the tissue sections. Typically, TCRBCL usually presents with large, widely scattered, neoplastic B-lymphocytes accompanied by a heavy infiltrate of reactive T-lymphocytes. 16 The neoplastic B-lymphocytes may appear as immunoblasts, multilobulated cells, or Reed-Sternberg-like cells in neoplasms from human beings. 17 Cellular atypia also has been observed in the TCRBCL reported in a cat. 16 The larger B-cells in the skin lesions of this cockatoo sometimes appeared immunoblastic, but generally lacked bizarre morphology. In human beings, TCRBCL may present as nodular neoplasms involving the lymph nodes; presentation as primary cutaneous lymphoma is unusual. 18 In contrast to humans, birds do not have classical lymph nodes; therefore, skin involvement ultimately may prove to be more common.
Although lymphoma is seldom reported in psittacine birds (compared to poultry), generalized visceral neoplasms are observed most commonly. 4-6,8-12,14 The present case is unusual in that numerous cutaneous nodules were present on initial examination; a gross appearance similar to that observed in chickens with the cutaneous form of Marek’s disease. Microscopically, the epidermotropism associated with cutaneous Marek’s disease was not observed in skin sections from this cockatoo. Primary cutaneous lymphoma also has been conclusively documented in a cockatiel with a localized mass on the forehead. 8 Although some articles suggest that cutaneous lymphoma occurs more frequently in psittacine birds, documentation of these assertions is lacking in the published literature. 8,10,14
The presence of a leukemic blood profile in this cockatoo is unusual. Generally, CBC counts have not been obtained or reported for most psittacine birds with lymphoma. Normal CBC counts were observed in three psittacine birds with lymphoma. 8,9 A leukemic blood profile has only been reported in a 23-year-old Amazon Parrot with a slowly growing periorbital mass on the right side of the face. 7 During clinical progression of the disease, the lymphocyte count declined from 49,500 cells per microliter to 5,100 cells per microliter over a 32 week observation and treatment period. Approximately 60% of the lymphocytes in blood smears from this bird had visible pseudopodia.
Interestingly, the lymphocytosis in the Umbrella Cockatoo of this report was primarily due to a reactive response of differentiated T-lymphocytes. The large-sized lymphocytes and lymphoblasts were B-cell lineage and were responsible for the leukemic blood profile. Some of these latter lymphocytes also had blunt pseudopods similar to those described in the Amazon Parrot.
Lymphoma in chickens commonly is associated with retrovirus or herpesvirus infection. In the present case we were unable to confirm herpesvirus infection by DNA in situ hybridization. Transmission electron microscopy failed to disclose either herpesvirus or retrovirus particles; however, formalin fixation artifacts precluded detailed examination of these tissue specimens. In a recently published report of a scarlet macaw with plasmacytoid lymphoma, another researcher also failed to demonstrate retroviral sequences in neoplastic tissue by polymerase chain reaction (PCR) amplification. 13
Sources and Manufacturers
a. Monoclonal mouse anti-human B lymphocyte antigen 36 kD (BLA.36) antibody, DAKO Corp. , Carpinteria, CA (USA).
b. Rabbit anti-human T cell, CD3 antibody, DAKO Corp. , Carpinteria, CA (USA). References
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