Extragonadal teratoma in a domestic turkey (Meleagris gallopavo domestica)

Abstract

This is the first report of a primary, spontaneous and, most probably, congenital teratoma in a domestic turkey, localized in front of the left eyeball. The unique localization allowed surgical excision of the tumour. The histopathological examination revealed that the tumour included structures derived from all three germ cell layers: ectoderm, mesoderm and endoderm (e.g. cartilaginous, osseous, haematopoietic, fibrous, nervous, glandular, squamous epithelial and smooth muscle tissues). The presence of epithelial cells as well as smooth muscle cells was confirmed using anti-cytokeratin and anti-desmin antibodies, respectively. The proliferative activity of the tumour cells was confirmed using proliferating cell nuclear antigen immunostaining. The other cases of teratoma in wild and domestic birds are reviewed briefly.

Introduction

Teratoma arises from totipotential germ cells that have undergone somatic differentiation and have given rise to two or more of the embryonic layers (endoderm, mesoderm and ectoderm), with a variety of tissues present (Kennedy et al., 1998). Some researchers presumed that teratoma arises from non-germinal cells of the early conceptus, stem cells, extra-embryonic cells, “included” twins or various cell types, depending on the anatomic site (Schelling, 1994 after Gonzalez-Crussi, 1982). The mechanism of development of the teratoma is unclear; the “misinvolvement–misenfoldment” hypothesis assumed that the disturbances in cell migration during the early embryonic stage of development might lead to intracranial teratoma (Sano, 2001). Histologically, teratomas are classified as: mature teratomas, characterized by differentiated tissues; immature teratomas, composed of immature but non-malignant tissues; and malignant teratomas (Lo Curto et al., 2007). Spontaneous teratoma is only rarely reported in domestic fowl and sporadically reported in wild avian species. All available reports describe teratomas diagnosed post-mortem. This is the first report of teratoma in a domestic turkey (Meleagris gallopavo domestica), diagnosed in an excisional biopsy specimen from the area of the left eye.

Materials and Methods

Case history

A 6-week-old turkey poult (male) was presented for clinical examination with a large excrescence in the area of the left eye (Figure 1). The general condition and behaviour of the bird were unaffected. Despite moderate scratching, the excrescence seemed to have no influence on the bird's comfort. After 2 days of clinical observation, surgical excision (using ketamine/xylazine intravenous anaesthesia) of the excrescence was conducted. Intrasurgical observation revealed that the excrescence was a tumour situated rostral to and loosely attached to the eyeball. The tumour was poorly encapsulated and well vascularized. The left eyeball was excised as well. After excision, the skin was sutured with a gauze swab inside the empty orbit. The swab was changed every day during the period of convalescence, which lasted 24 days.

Figure 1.  The large excrescence in front of the left eyeball in a turkey poult.

Macroscopically, the tumour was heterogeneous and partially covered with crusts (this area protruded outwards). The cut surface was pale and yellowish with visible aggregates of melanin. The consistency was firm, and the structure was granular and cystic.

Histopathology and immunohistochemistry

The tumour was surgically excised, and the samples were fixed in 10% buffered formalin, embedded in paraffin, cut at 5 µm and mounted. The sections were processed routinely and stained with Alcian blue-PAS (Bancroft & Gamble, 2008), as well as with haematoxylin and eosin, Mallory trichrome (staining kit; Bio-Optica, Milano, Italy) and cresyl violet, according to conventionally accepted procedures (Bagiński, 1969). Immunohistochemical examination was performed using anti-cytokeratin (1:50 dilution, clone AE1/AE3; DAKO, Carpinteria, California, USA), anti-proliferating cell nuclear antigen (1:200 dilution, clone PC10; DAKO, Glostrup, Denmark) and anti-desmin (1:50 dilution, clone D33; DAKO, Glostrup, Denmark) monoclonal mouse anti-human antibodies. The primary antibody was detected using a system based on a horseradish peroxidase-labelled polymer conjugated with secondary antibodies (Impress Universal Reagent Anti Mouse/Rabbit Ig, VECTOR, Burlingame, California, USA). The antigen–antibody complexes were visualized using 3,3-diaminobenzidine (DAB) and a chromogen substrate (DAB Substrate Kit for Peroxidase; VECTOR, Burlingame, California, USA), which resulted in a brown precipitate at the antigen site. The specimens were counterstained with Mayer's haematoxylin (3 min), dehydrated and mounted using Canadian balm. For the negative control, primary antibody was replaced with Mouse IgG2a (1:200; DAKO, Glostrup, Denmark). The appropriate normal canine tissue sections were used as positive control. Each section was investigated using a Panoramic Scanner MIDI (3DHISTECH, Budapest, Hungary). Photographs were prepared using Panoramic Viewer software (3DHISTECH, Budapest, Hungary).

Results

The tumour comprised many different tissue components. The most apparent feature was the presence of the multiform cystic spaces lined by simple columnar epithelium with numerous goblet cells. The goblet cells contained acidic or neutral mucus, depending on their localization (Figure 2). These glandular structures formed crypts and simulated the intestine. The pseudostratified ciliated epithelium resembled the epithelium of the respiratory system. The hyaline cartilage formed islands of various sizes, scattered throughout the tumour mass. Some of these islands contained embryonic hyaline cartilage with an irregular chondrocyte arrangement. The chondrocytes of the embryonic hyaline cartilage did not form chondrones and concentrated in wreath-like structures in some areas (Figure 3). There were also areas of endochondral ossification (Figure 4). The trabecular bone cavities contained bone marrow, which consisted of heterophils and lymphoblasts. The glandular tissue was surrounded by smooth muscle cells in the form of thick strands. The melanocytes were organized in bands that resembled those of the ciliary body. The stratified squamous epithelium occasionally formed keratin pearls (Figure 5). Primitive nervous tissue formed rosettes with cystic spaces; these areas mimicked the neural tube. The areas of well-developed neurons (dispersed, with pale nuclei and distinct nucleoli) were well circumscribed and showed distinct angiogenesis. There were also small areas of ganglion cells surrounded by satellite glial cells.

Figure 2.  The glandular structures with numerous goblet cells (Alcian blue-PAS). Bar = 50 μm.

Figure 3.  The chondrocytes of the embryonic cartilage formed wreath-like structures (haematoxylin and eosin). Bar = 50 μm.

Cytokeratin immunoreactivity was positive in the cytoplasm of the epithelial cells (cuboidal cylindrical, pseudostratified, squamous) (Figure 6) and strongly positive in the horny pearls. The bundles of myocytes and the myoepithelial cells surrounding the glandular tissue were strongly immunoreactive for desmin (Figure 7). Proliferating cell nuclear antigen immunoreactivity was strong in the epithelial cells, myocytes, many of the chondrocytes (Figure 8), satellite glial cells and proliferating endothelial cells.

Figure 4.  The area of endochondral ossification (haematoxylin and eosin). Bar = 200 μm.

These components represent all three germ layers (Table 1). All tissues represented in the tumour were mature, except for poorly differentiated cells in the area resembling a neural tube.

Figure 5.  The stratified squamous epithelium formed a horny pearl (haematoxylin and eosin). Bar =50 μm.

Table 1.  Derivation of tissue components present in the teratoma.

Germ layer
Ectoderm	Mesoderm	Endoderm
Keratinized squamous epithelium	Smooth muscle	Columnar epithelium and the goblet cells
Nervous tissue	Cartilage	Pseudostratified epithelium
Melanocytes	Bone

The eye was approximately 15 to 18 mm in diameter, spherical, and loosely attached to the tumour mass. The surface was irregular and nodular. The tumour mass attached to the sclera did not penetrate the eyeball and displayed a structure similar to that of the main tumour mass. The retina displayed rod and cone layer dysplasia. Rods and cones were degenerated and arranged irregularly. The cornea displayed cutaneous metaplasia, with nodular protrusions filled with adipose tissue and covered with squamous epithelium that formed at the border of the cornea and sclera (corneal limbus). The squamous epithelium was dysplastic (displayed dysplastic cornification) and formed cystic spaces. The outer, cornified layer displayed massive exfoliation and was focally strongly infiltrated with heterophils. The ossicles normally present in the avian sclera were focally hyperplastic, and intramembranous ossification was observed focally. The Harderian gland consisted of columnar epithelial cells of foamy appearance, with strong secretory activity; individual cells demonstrated secretory retention with balloon enlargement. The gland was infiltrated by large numbers of heterophils.

Figure 6.  The epithelial cells displayed cytokeratin immunoreactivity (cytokeratin immunostaining). Bar = 50 μm.

Discussion

The most common location of avian teratoma is the ovary, testicle or coelom; there are few reports of avian teratomas situated retrobulbarly or intracranially (Table 2) and only one report of an avian teratoma on the head surface (Homer, 1991). No previous reports of extragonadal teratoma in turkeys were found. The origin of the teratoma described is uncertain, as it involved a large part of the cutis and subcutis of the head and was extending to the eyeball. The tumour was probably derived from misplaced embryonic cells. The teratomas in birds are in most cases benign, but due to their large size and location can often lead to death (Table 2). In the case reported here, the mature character of the tumour and its external location allowed surgical removal of the tumour and the affected eyeball.

Figure 7.  The bundles of myocytes showed strong desmin immunoreactivity (desmin immunostaining). Bar = 200 μm.

Table 2.  Reports of teratoma in wild and domestic avian species.

Species	Age	Sex	Localization	Reference
Lesser kestrel (Falco naumanni)	10 days old	Unknown	Retrobulbar, intracranial	Lopez & Múrcia (2008)
Fantail pigeon (Columba livia domestica)	1 year old	Female	Cerebrum	Hooper (2008)
Bald eagle (Haliaeetus leucocephalus)	1 year old	Male	Coelom	Ford et al. (2006)
Eurasian buzzard (Buteo buteo)	10 years old	Male	Coelom	Lopez & Múrcia (2008) after Forbes et al. (2000)
Great blue heron (Ardea herodias)	Adult	Female	Retrobulbar	Schelling (1994)
Domestic goose (Anser anser domesticus) (2 cases)	Adult/unknown	Male/unknown	Abdomen, ovary	Reece & Lister (1993), Rigdon (1967)
Budgerigar (Melopsittacus undulates) (5 cases)	Unknown	Male	Testes	Neumann et al. (1986)
Black-headed gull (Chroicocephalus ridibundus)	Adult	Female	Multi-organ	Baker & Chandler (1981)
Seychelles kestrel (Falco araea)	30 days old	Unknown	Body surface: head and breast	Cooper et al. (1978)
Domestic fowl (Gallus gallus domesticus) (42 cases)	4 weeks old to 3 years old (mostly young birds)	67.6% males	Abdomen, head of merkel diverticulum, kidney, ovary, testis, pineal gland, adrenal gland, spinal cord, lungs, cerebellum, retroocular, mesentery	Sugiyama et al. (1983), Gupta (1976), Helmboldt et al. (1974), Campbell & Appleby (1966), Feldman & Olson (1959), Jones (1964), Campbell (1951, 1962), Mashar (1932)
Pekin duck (Anas platyrhynchos domestica), Muscovy duck (Cairina moschata); hybrids (11 cases)	5 months old to 2 years old (mostly young birds)	50% males	Thorax, abdomen, subcutis and parietal bone, pons, testis, gonad (in hybrid)	Mutinelli & Vascellari (2002), Bolte & Burkhardt (2000), Homer & Riggs (1991), Cullen et al. (1991), Hamir (1985), Rigdon & Leibovitz (1970), Rigdon (1967), Mashar (1932)

Immunohistochemistry examination was previously used in diagnosing avian teratoma, using vimentin, smooth muscle actin, pancytokeratin and neuron-specific enolase (Hooper, 2008). In this study, the combination of histochemical and immunohistochemical stainings allowed identification and description of all of the tissue components. The proliferating cell nuclear antigen immunoreactivity of most of the tissue components of the teratoma suggests that the tumour had the capacity for growth.

Figure 8.  The proliferating chondrocytes of the embryonic cartilage (proliferating cell nuclear antigen immunostaining). Bar = 50 μm.