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Malignant lymphoma of T-cell origin in a Humboldt penguin (Spheniscus humboldti) and a pink-backed pelican.

Abstract: Multicentric T-cell lymphomas were diagnosed in two birds from separate zoological collections: one in a 27-year-old female Humboldt penguin (Spheniscus hurnboldti) and the second in an adult pink-backed pelican (Pelecanus rufescens). The main clinical sign in the penguin was dysphagia caused by lymphoma formation in the esophagus. Besides the esophageal lymphoma, neoplastic lymphoid cells were observed in the adrenal glands, liver, kidneys, lung, proventriculus, and gizzard. The pelican was found dead without a clinical history. Neoplastic lymphoid cells were observed in the kidneys, liver, pancreas, spleen, ventriculus, and small intestine. Neoplastic cells of the penguin as well as of the pelican were immunoreactive to CD3 antigen, suggesting the lymphomas were of T-cell origin. In both cases, test results were negative for Marek's disease virus, avian leukosis virus, and reticuloendotheliosis virus. In the pelican, a skin melanoma was diagnosed on the left throat pouch in addition to the multicentric T-cell lymphoma.

Key words. T-cell lymphoma, CD3 antigen, avian, melanoma, penguin, Spheniscus humboldti, pelican, Pelecanus rufescens

Clinical Report

A 27-year-old female Humboldt penguin (Spheniscus humboldti) from a zoo collection presented with a 3-week-history of dysphagia. It was part of a group of penguins that were kept in an outside as well as indoor enclosure throughout the year. The diet consisted of herrings, which were supplemented with one fish-eater tablet each (Mazuri Zoo Food, aleckwa Tiernahrung, Altrip, Germany) per week. A nodular solid mass was palpable in the cranial esophagus. Dimension of the mass was 6 x 3 x 1 cm. The penguin was anesthetized, and stones, straw, and fish particles were removed by endoscopy. The bird was treated with enrofloxacin (Baytril, Bayer, Leverkusen, Germany) and meloxicam (Metacam, Boehringer Ingelheim Vetmedica, Ingelheim, Germany) for a working diagnosis of a granulomatous esophagitis caused by a foreign body. Three weeks later, a second endoscopy showed that the surface of the esophagus in the damaged region was covered by whitish-grey deposits that almost completely occluded the esophageal lumen. The penguin died shortly after the second endoscopy.

An adult female pink-backed pelican (Pelecanus rufescens) was found dead without preceding clinical symptoms. The pelican was from a private zoological collection and was part of a group of pelicans that were kept outside throughout the year on a nonfenced lawn together with greater flamingos (Phoenicopterus roseus), saddle-bill storks (Ephippiorynchus senegalensis), and Bewick's swans (Cygnus colombianus bewickii). The pelicans were fed whole fish (5 8 kg/d) bought at local markets every day. The diet was supplemented with two fish-eater tablets per fish (Mazuri Zoo Food) per week.

[FIGURE 1 OMITTED]

Necropsies were performed in both cases immediately after the birds were found dead according to standard procedures. (1) Samples were taken from both birds for bacteriologic, parasitologic, cytologic, and histopathologic examination and processed according to standard protocols as described elsewhere. (2) At necropsy, the penguin was emaciated, weighing 2.9 kg. A white, firm intramural mass, 8 x 4 x 1 cm, was present in the oral third segment of the esophagus. The mass completely blocked the lumen (Fig 1). The liver and kidneys contained multifocal, white firm nodules of 0.2 cm diameter. The lungs were diffusely blackish-red, and the small intestine was filled with black watery contents. No gross pathologic findings were detected in the other organs. Results of cytologic examination of the esophageal mass revealed heterophils, lymphocytes, and macrophages, as well as numerous bacterial rods and cocci. The pelican's weight at postmortem examination was 3.5 kg, and its body condition was poor. A line of black spots was detected under the skin on the left throat pouch (Fig 2). The spleen, liver, kidneys, and pancreas were enlarged with white spots of 0.2 cm diameter. The intestine contained a blackish-red watery content. No gross pathologic findings were seen in other organs. Lymphocytes were observed on cytologic examination of imprints of liver, spleen, and lungs. No parasites were detected in the intestines of either bird. Results of bacterial and fungal culture of samples from all organs were negative, except for those from the esophageal mass from the penguin, which revealed Streptococcus species, Staphylococcus species, and Escherichia coli.

[FIGURE 2 OMITTED]

In the penguin, histologic examination revealed a marked infiltration of pleomorphic neoplastic lymphocytes in the kidneys, liver, lungs, adrenal glands, esophagus, proventriculus, and gizzard. The neoplastic cells had round to oval hypochromatic nuclei with numerous nucleoli, small to moderate amounts of cytoplasm, and distinct cell borders. Numerous mitotic figures and single apoptotic cells were present (Fig 3a). Additionally, a small amount of urate tophi and degenerated tubular epithelial cells were seen in the kidneys. In the pelican, the kidneys, liver, pancreas, spleen, gizzard, and small intestines were infiltrated multifocally by neoplastic lymphocytes. The neoplastic lymphocytes had round hypochromatic nuclei, small amounts of cytoplasm, and distinct cell borders. Single mitotic figures and single apoptotic cells were present (Fig 4a). In the pelican, a subcutaneous focus of neoplastic melanocytes additionally was seen (Fig 5). Neoplastic cells were predominantly of epitheloid shape and moderately pleomorphic. The cells had oval hypochromatic nuclei and moderate to large amounts of markedly pigmented cytoplasm. Bleaching process resulted in loss of coloration, and single mitotic figures were detectable. The neoplastic growth was expansive.

[FIGURE 3 OMITTED]

The peroxidase anti-peroxidase (PAP) technique was used to identify CD3, CD45, and CD79 antigens in tissue sections. Serial sections were dewaxed, rehydrated, and treated with hydrogen peroxide 0.5% in methanol for 30 minutes at room temperature to eliminate endogenous peroxidase activity. CD45 and CD79 were detected with citrate pretreatment (with 10 mM citrate buffer at pH 6.9 for 30 minutes at 96[degrees]C), whereas CD3 detection required protease predigestion (0.05% protease XXIV; 37[degrees]C; 5 minutes; P8038, Sigma-Aldrich Chemie GmbH, Munich, Taufkirchen D82024, Germany). When using polyclonal antibody as the primary antibody, a blocking step with 50% pig serum in Tris-buffered saline (TBS) at room temperature was applied to "the sections for 10 minutes. Slides were treated with the following primary antibodies diluted in TBS at 4[degrees]C overnight: rabbit anti-CD3 at a 1:200 dilution in TBS (A 0452, DAKO GmbH, Hamburg D-22769, Germany), mouse anti-CD45 at a 1:1000 dilution in TBS (B 220, Camon, Wiesbaden D-65205, Germany), and mouse anti-CD79 at a 1:10 dilution in TBS (M 7051, DAKO). Secondary antibodies were rat anti-mouse IgG (1:100; 415-005-166, Dianova GmbH, Hamburg D-20354, Germany) or swine anti-rabbit IgG (1:100; Z0196, DAKO). Subsequently mouse PAP complex (1:500; 223005025, Dianova) or rabbit PAP complex (1:100; Z0113, DAKO) were used. Diaminobenzidinetetrahydrochloride (Fluka Chemie GmbH, Buchs, Switzerland) was used as chromogen, and slides were counterstained with Papanicolaou solution (Merck GmbH, Darmstadt, Germany). Primary antibody was replaced by normal mouse (X0931, DAKO) or rabbit (X0930, DAKO) serum to establish negative control tissue sections. An expression of CD3 was detected immunohistochemically on lymphocytes in their T-cell areas (gut and spleen). Furthermore, the neoplastic lymphocytes in both animals intensively expressed CD3 (Figs 3b and 4b). However, results of CD45 and CD79 immunolabeling were negative in the neoplastic lymphocytes as well as in the T-cell areas (gut and spleen), suggesting that these antibodies were not cross-reacting in this species.

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

The diagnosis in both birds was malignant lymphoma of T-cell origin.

Samples from neoplastic tissue (esophagus and liver from the penguin, liver and spleen from the pelican) were homogenized and passed through 0.45-[micro]m mesh filters. The chorioallantoic cavities of pathogen-free chicken embryos (Lohmann Valo SPF, Lohmann Tierzucht GmbH, Cuxhaven, Germany) were inoculated at day 10 of incubation. After 7 days of incubation, the chorioallantoic fluid was harvested and passaged into fresh embryos for a total of 3 passages. Additionally, monolayer cultures of primary specific pathogen--free chicken embryo liver cells were inoculated and passaged 1 time after 7 days of incubation.

The DNA was extracted from tissue samples with the use of spin columns (NucleoSpin Tissue, Macherey-Nagel GmbH & Co. KG, Duren, Germany). Polymerase chain reaction testing for DNA of reticuloendotheliosis virus was performed as described. (3) For the detection of DNA of Marek's disease virus (serotype 1), a real-time PCR protocol was used. (4) No viruses were cultivated from the different organs, and neither DNA of gallid herpesvirus-1 nor of reticuloendotheliosis virus was amplifiable in liver, spleen, or kidneys of either bird.

Discussion

Multicentric lymphoma was diagnosed in a Humboldt penguin and a pink-backed pelican. The T-cell origin was proven immunohistochemically by positive reaction to CD3 antigen. To our knowledge, this is the first report of a disseminated T-cell lymphoma in both species. The pan T-cell marker CD3 used in these cases is broadly reactive among vertebrate taxa. In these 2 birds, viral pathogens were not detected by virologic or molecular methods; therefore, a nonviral cause for the lymphomas was considered likely.

Reports of neoplastic diseases in penguins and pelicans are rare. Two cases each of melanoma, cholangicarcinoma, ventricular adenocarcinoma, and lymphocytic sarcoma/mixed cell lymphoma and 1 case of choanal squamous cell carcinoma have been described in penguins. (5-14) Neoplastic diseases that have been reported in pelicans are 8 cases of chondrosarcoma, 3 cases of squamous cell carcinoma, and 1 case each of cholangiocarcinoma, bronchial carcinoma, phaeochromocytoma, and ganglioneuroma. (7,15-17)

Lymphomas of T-cell origin confirmed by positive immunoreactions to CD3 antigens have been described in Amazon parrots (Amazona species) and a great horned owl (Bubo virginianus). (18-21) Lymphoma in birds can be spontaneous or virus induced. In gallinaceous poultry, Marek's disease virus is able to induce T-cell lymphoma. (22-26) Avian leukosis virus causes B-cell lymphomas mainly in gallinaceous poultry, and reticuloendotheliosis virus has been associated with either B- or T-cell lymphomas in gallinaceous birds, geese, and a European starling (Sturnus vulgaris). (27-29) Marek's disease virus, avian leukosis virus, and reticuloendotheliosis virus have not been described in penguins or pelicans.

The cause of death in the penguin was probably dehydration and hypoglycemia as a result of the esophageal obstruction. The lymphoma entirely blocked the esophageal lumen, leaving the bird unable to eat or drink. The renal failure with formation of gout tophi and degeneration of the renal tubules indicated severe dehydration. Hemorrhagic diathesis in the gut is a consequence of hypoglycemia, which most likely was the cause of death in the pelican. (30)

In addition to the lymphoma, a melanoma was diagnosed in the subcutaneous tissue of the pelican. Circumscription as well as differentiation of melanocytes in this melanoma was good, and mitotic figures were rare. No metastases were detected in other body regions. An etiological connection between both neoplasms is unlikely.

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Volker Schmidt, Dr MedVet, Dipl ECZM (Avian), Hans-Christian Philipp, Dr MedVet, Jens Thielebein, MedVet, Susanne Troll, MedVet, Christiana Hebel, MedVet, and Heike Aupperle, Dr habil MedVet

From the Clinic for Birds and Reptiles (Schmidt), and Institute of Pathology (Aupperle), Faculty of Veterinary Medicine, University of Leipzig, An den Tierkliniken 17, D-04103 Leipzig, Germany; Lohmann Tierzucht GmbH, Abschnede 64, D-27472 Cuxhaven, Germany (Philipp), the Institut ftir Agrar- und Ernahrungswissenschaften, Martin-Luther-Universitat Halle-Wittenberg, Emil-Abderhalden Strasse 28, D-06108 Halle, Germany (Thielebein, Troll), and Al Wabra Wildlife Preservation, PO Box 44069 Doha, State of Qatar (Hebel).
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Title Annotation:Pelecanus rufescens
Author:Schmidt, Volker; Philipp, Hans-Christian; Thielebein, Jens; Troll, Susanne; Hebel, Christiana; Auppe
Publication:Journal of Avian Medicine and Surgery
Article Type:Report
Geographic Code:4EUGE
Date:Jun 1, 2012
Words:2645
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