Lymphoma in 2 black vultures

Abstract

Vultures have suffered a drastic population decline mainly due to poisoning and traumatic lesions; neoplastic diseases in these birds are rarely documented. Here we describe the clinical and pathologic findings of lymphoma in 2 free-ranging black vultures (Coragyps atratus). Upon initial evaluation, both birds were severely emaciated; vulture 1 had proptosis of the right eye and vulture 2 swelling of the left wing. The vultures died shortly after presentation and were autopsied. In both birds, the thymus and many other organs were expanded by poorly demarcated, white, soft masses that were composed histologically of proliferating lymphocytes of monomorphic appearance. In vulture 2, thickening of the left-wing bones appeared to be caused by periosteal reaction, associated with bone invasion by the same type of lymphocytes, and granulomatous osteomyelitis. By immunohistochemistry, neoplastic cells were reactive for CD3 and negative for PAX5. The final diagnoses were multicentric T-cell lymphoma. PCR assays for Marek disease, avian leukosis, reticuloendotheliosis, and bovine leukemia viruses were negative in both cases. To our knowledge, lymphoma has not been reported previously in vultures.

Vultures comprise 23 species (16 Old World and 7 New World) of scavengers that play an essential role in ecosystems, but have suffered a dramatic global population decline over the past few decades. ⁸ Currently, 13 species are classified as vulnerable, endangered, or critically endangered. ⁷ The primary causes of mortality for these birds include toxins (mainly pesticides, diclofenac, or lead) and traumatic lesions (mainly electrocution or gunshot); neoplastic diseases are rarely reported. ⁸ A search of Google, PubMed, Web of Science, and Scopus retrieved only 2 reports describing an uncharacterized malignant round-cell neoplasm in a California condor (Gymnogyps californianus) and a pulmonary carcinoma in a lappet-faced vulture (Torgos tracheliotos).^17,19 A review of postmortem vulture submissions from the Departments of Veterinary Pathology at the Federal University of Rio Grande do Sul (UFRGS; Porto Alegre, RS, Brazil; 2010–2023) and Pathobiology at the University of Guelph (Guelph, ON, Canada; 2007–2023) revealed only 2 cases of neoplasia (both lymphomas) among 36 autopsied vultures (Suppl. Table 1). Here we describe the clinical and pathologic findings of these lymphomas in 2 black vultures (Coragyps atratus).

Two free-ranging adult male black vultures were found emaciated in the courtyards of different rural houses (Viamão, RS, Brazil) within a 10-y period. Both vultures were referred for examination to the Center for Conservation and Rehabilitation of Wild Animals at the UFRGS. Vulture 1 was presented with proptosis of the right eye, which was enucleated without complications and fixed in 10% neutral-buffered formalin for histopathology. The patient died suddenly the following morning. Vulture 2 had difficulty flying and marked swelling of the left wing, the bones of which were thickened and hard upon palpation. Radiographs revealed multifocal-to-diffuse periosteal hyperostosis of the left humerus, radius, ulna, metacarpus, and basal phalanx (Fig. 1), along with areas suggestive of bone lysis. Despite supportive care, the patient died suddenly 3 d later. Both vultures were immediately submitted for autopsy.

Figures 1–5.

Lymphoma in 2 black vultures. Figure 1. Multifocal-to-diffuse periosteal hyperostosis expands the cortical surface of the left-wing bones; case 2. Figure 2. White masses infiltrate the thymus (asterisk), trachea, and heart; case 1. Figure 3. The liver is enlarged, and white masses infiltrate the cranial celomic surface (asterisk) and heart; case 2. Figure 4. Disseminated white masses expand the kidneys; case 2. Figure 5. Macerated and bleached bones of the left wing highlight the irregular, porous surface; case 2.

The main gross finding in vulture 1 was a 6 × 3 × 3-cm, white, soft, poorly delimited mass that effaced the thymus in the cervical region and extended into the cranial celom (Fig. 2), infiltrating the adjacent trachea and the breast muscle. Internally, the right ocular globe was diffusely effaced by a white, soft mass. The epicardium had a 1-cm, white, slightly raised, soft plaque, and the wall of the proventriculus was diffusely thickened and discolored on cross-section. Similarly, vulture 2 had an 8 × 4 × 3.5-cm, white, soft, poorly delimited mass that effaced the thymus in the cervical region and extended into the cranial celom, infiltrating the serosa of lung, heart, and trachea. Several muscles in the pectoral girdle (pectoralis major, supracoracoideus) and left wing (biceps branchii, triceps branchii, ectepicondylo ulnaris, pronator superficialis, extensor carpi radialis) were also infiltrated. Additional findings included hepatomegaly (Fig. 3), splenomegaly, and expansion of both kidneys by coalescing, ~1.0-cm, white, soft masses (Fig. 4). Some affected bones from the left wing of vulture 2 were submitted for cold water bacterial maceration and bleached by immersion in 10% hydrogen peroxide. The maceration process highlighted the expansion of the cortical profile by irregular bony proliferations (Fig. 5).

Histologic evaluation of H&E-stained tissue sections from both cases revealed effacement of multiple tissues by non-encapsulated, infiltrative, densely cellular sheets of monomorphic lymphocytes (Fig. 6). Neoplastic cells were up to an erythrocyte in diameter and had scant eosinophilic cytoplasm with distinct margins. The nuclei were round, large, occasionally eccentric, and had coarse-to-condensed chromatin and 1 nucleolus. Anisokaryosis and anisocytosis were moderate. There were 9 (case 1) and 15 (case 2) mitotic figures in 2.37 mm² (10 FN22/40× fields), and occasional single-cell necrosis. In vulture 1, neoplastic lymphocytes infiltrated the breast muscle, epicardium, proventriculus, liver, kidneys, pancreas, small intestine, right eye, and leptomeninges. In vulture 2, neoplastic lymphocytes infiltrated the skeletal muscles of the pectoral girdle and left wing, bones of the left wing, liver, spleen, heart, lung, kidneys, adrenal glands, air sacs, brain, and trachea.

Figures 6–9.

Lymphoma in 2 black vultures. Figure 6. Sheets of neoplastic lymphocytes efface the renal interstitium; case 1. H&E. Figure 7. Proliferation of lamellar bone expands the periosteum of the left basal phalanx. Neoplastic lymphocytes infiltrate the proliferated bone; case 2. H&E. Figure 8. Medullary cavity of the left basal phalanx affected by coagulative necrosis with granulomatous inflammation and infiltrated by neoplastic cells; case 2. H&E. Figure 9. Neoplastic lymphocytes have strong membranous immunolabeling for CD3; case 2. Immunohistochemistry.

The bones of the left wing in vulture 2 had circumferential proliferation of lamellar bone perpendicular to the cortical surface, consistent with periosteal reaction. In some areas, the pre-existing cortical bone was lost, and the periosteal new bone and medullary cavity were infiltrated by neoplastic cells (Fig. 7). Additionally, occasional areas of coagulative necrosis surrounded by epithelioid macrophages and multinucleate cells were observed in the medullary cavity of the left basal phalanx (Fig. 8). No organisms were identified with Gram, Grocott methenamine silver, and Ziehl–Neelsen stains.

To identify the immunophenotype of the neoplastic cells, tumor sections from both cases were tested by immunohistochemistry to evaluate expression of CD3 (polyclonal rabbit antibody, diluted 1:1, cat. 0452; Dako) and PAX5 (monoclonal mouse antibody, clone DAK-Pax5, ready-to-use, cat. GA650; Dako) using automated stainers. After deparaffinization, the slides were submitted to heat epitope retrieval with target retrieval solution (Dako) for 30 min at 97°C (CD3) or Bond epitope retrieval solution 2 (Leica) for 20 min at 100°C (PAX5). EnVision FLEX/HRP (Dako; CD3) and Bond polymer refine detection (Leica; PAX5) were used as the detection systems. After visualization of the reaction with 3,3′-diaminobenzidine, sections were counterstained with hematoxylin. Normal spleen and thymus from another black vulture were used as external positive controls. As a negative control, tumor sections were incubated with polymer negative control serum (Biocare Medical). Nearly 90% of the neoplastic cells from both vultures had widespread, moderate (case 1) to strong (case 2) membranous and, to a lesser extent, cytoplasmic immunolabeling for CD3 (Fig. 9). There was no immunolabeling for PAX5. Both external (Suppl. Figs. 1–4) and internal positive controls reacted as expected, indicating appropriate cross-reactivity of the antibodies with this taxon. Based on the gross and microscopic findings, both birds were diagnosed with multicentric T-cell lymphoma.

Although lymphomas in non-poultry species are considered mainly sporadic with unknown etiology, ³ Marek disease virus (MDV; family Orthoherpesviridae, taxon species Mardivirus gallidalpha2), reticuloendotheliosis virus (REV; Retroviridae, Gammaretrovirus aviretend), and avian leukosis virus (ALV; Retroviridae, Alpharetrovirus avileu) are oncogenic viruses known to cause round-cell tumors in poultry, particularly chickens. ²⁰ Experimental and rare putative natural infections with these viruses have been reported to induce cell transformation and lymphoma in avian species taxonomically distant from chickens.^6,12 Therefore, fresh tissues from the thymic masses from both vultures were submitted to Simbios Biotecnologia (Cachoeirinha, RS, Brazil) to test for the presence of MDV, REV (provirus), and ALV (provirus) DNA by quantitative PCR, according to proprietary methods. Thymic masses from both vultures were also tested for bovine leukemia virus (BLV; Retroviridae, Deltaretrovirus bovleu), which can induce round-cell tumors in experimentally infected chickens. ¹ The proviral DNA was extracted from formalin-fixed paraffin-embedded (case 1) and fresh (case 2) tissues using a standard phenol–chloroform protocol, and PCR or nested PCR assays were performed to detect the presence of BLV env, gag, pol, and tax genes. ² All samples tested negative (data not shown).

Lymphoma is a heterogeneous group of malignancies with proliferations of lymphocytes and is considered the most common hemolymphatic neoplasm in birds.^13,16,22 These neoplasms have been reported in several species, including Psittaciformes, Galliformes, Passeriformes, Strigiformes, Columbiformes, and Anseriformes.^5,15,16 Lymphoma in birds tends to be multicentric with variable distribution and infiltrative growth in various organs, often without clear evidence of the tissue of origin.^5,16 The organ systems most frequently affected include hepatic, gastrointestinal, urogenital, lymphoid, and respiratory systems. ⁵ Single-organ involvement is less frequent, although it has been described in the skin, liver, and brain. ⁵ In our 2 cases, many organs were affected, but the extensive involvement of the thymus suggested a thymic origin. Although the thymus is composed primarily of T lymphocytes, both T- and B-cell thymic lymphomas can occur in domestic animals, and affected individuals are often young. ²² However, thymic lymphomas in birds are typically of T-cell origin and affect adult animals,^9,23 consistent with our cases.

In domestic mammals, lymphomas are classified according to distinct morphologic, immunophenotypic, molecular, and biological characteristics, which can directly impact the prognosis. ²² Similar classification systems are rarely applied to wild and pet birds, mainly because these tumors are uncommon in many avian species and prognostic data are limited.^13,18 A retrospective study using morphologic and immunophenotypic characterization revealed that most lymphomas in psittacine birds are of B-cell origin, predominantly affecting the gastrointestinal and urogenital systems; T-cell lymphomas are more commonly associated with the respiratory system. ⁵ Moreover, diagnosing lymphomas in birds can be further complicated by limited information on the cross-reactivity of avian tissues with antibodies developed for mammals. ⁵ Some studies have reported inconsistent results using lymphocyte markers in birds.^10,11,21 In our study, both CD3 and PAX5 antibodies were successfully validated using internal and external positive controls from a conspecific, demonstrating the utility of these lymphocyte markers in black vultures.

The excessive bone formation (hyperostosis) seen in vulture 2 was unique. Hyperostosis is considered a nonspecific reaction that can occur in response to different bone lesions, such as metabolic, traumatic, and neoplastic diseases. ⁴ Based on radiographic and histopathologic findings in vulture 2, we deemed the bone reaction to be secondary to either direct lymphomatous infiltration or a primary granulomatous osteomyelitis of unspecified cause. In fact, the few sections of bone available from the left wing (most of the bones were used for maceration) had granulomatous inflammation that could have directly caused periosteal reaction and bone lysis. After maceration, sufficient tissue to attempt bacterial isolation was not available, and infectious agents were not identified by histology. Therefore, it is challenging to determine the relative contribution of osteomyelitis and lymphoma to the bone changes observed in the left wing of vulture 2. Clinical differential diagnoses for the periosteal new bone formation also included hypertrophic osteopathy and osteopetrosis. Lack of bilateral symmetric lesions, negative molecular results for ALV (the causative agent of osteopetrosis in chickens), ¹⁴ and focal disease identified on the left wing made these differentials less likely.