I. Comp. Path. 1991 Vol. 105

Salivary Gland Oncocytes in African Hedgehogs ( A te l e r ix a l b i v e n t r i s ) Mimicking Cytomegalic

Inclusion Disease

S. R. Brunnert*, G. T. Hensley*, S. B. Citino~, A. J. Herron* and N. H. Altman*

*Division of Comparative Pathology, Department of Palhology, University of Miami School of Medicine, P.O. Box 016960, Miami, FL 33101 and t Department of Veterinary Sciences,

Miami Melrozoo, Miami, FL 33177, U.S.A.

Summary The salivary glands from three African hedgehogs contained multiple li)ci of cytomegallc cells, which occasionally had a mild to moderate infiltrate of lymphocytes at the periphery. The cytomegalic cells were 35 to 40bun in diameter with abundant aeidophilic granular to hyalin cytoplasm. The nuclei were enlarged with clumped marginalized chromatin and a large, (6 to 8 I.tm in diameter) central, brightly eosinophilic nucleolus that had the appearance of an inclusion body by light microscopy. Histochemically most of the cytolaegalic ceils contained cytoplasmic metachromatic granules with Feyrter's thionine inclusion stain. Scattered cells at the periphery of the cytomegalic loci contained periodic acid-Schiff-positive cytoplasmic granules. Ultrastructurally the cytornegalic cells contained numerous tightly-packed, often bizarre, enlarged mitochondria that completely filled the cytoplasm. The nucleus consisted of a dense central core of chromatin associated with the nucleolus and the remaining chromatin was clumped and marginalized. Nuclear and cytoplasmic virions consistent with cytomegalovirus were not present. Histochemical stains of the nucleus for heavy metals were negative. The ultrastructural and hlstochemical findings of the cytomegalic cells were consistent with oncocytes. Previous reports in the literature of similar cells in the salivary glands of insectivores appear to have been erroneously described as cytomegalovirus infections.

Introduction

African hedgehogs (Atelerix albiventris) are classified in the order Insectivora which contains approximately 400 species of small mammals occurring throughout the world. Hedgehogs belong to the superfamily Erinaceoidea with species present in Europe, Asia and Africa. Through the process of evolution, both primates and rodents apparently originated from insectivores and then evolved separately. Because of their close relationship with primates, the anatomical and physiological features of insectivores have been well documented. However, morphological descriptions of normal organ histology and insectivore diseases are infrequent in the literature (Stunkard, Migaki, Robinson and Christian, 1975; Cosgrove, 1986; Griner, 1983).

0021-9975/91/050083 + 09 $03.00/0 © 1991 Academic Press Limited

84 s .R . Brunnert e t al.

The cytomegaloviruses are included in the herpesvirus group and can infect a variety of animal species (Hanshaw, 1968). Cytomegalic inclusion disease has been reported in the salivary gland of several species of insectivores, including five African hedgehogs (Karstad, 1975), a variety of shrews (Cosgrove, 1986; Mineda, 1981), and moles (Mineda, 1981). In one report, ul trastructural studies revealed scattered single ductular cells with cytoplasmic and nuclear virions consistent with cytomegalovirus (Mineda, 1981). In the remaining reports, the diagnosis of cytomegalovirus was based entirely on the light microscopic finding of clusters of cytomegalic cells with intranuclear inclusions limited to the salivary glands (Cosgrove, 1986; Karstad, 1975).

Oxyphil cells (oncocytes) are normally found in the parathyroid of a variety of mammals, but are absent in the rat, chicken and many species of lower animals (Capen, 1983). In man, they normally occur singly or in small clusters in numerous organs (Hamperl, 1962b), increase with age (Tandler, 1966; Hamperl, 1962b), different disease processes (Hamperl, 1962b; Allen and Thorburn, 1981; Matsuta, 1982) and can be the main cellular component of neoplasms (Hamperl, 1962b; McGregor, Lotuaco, Rao and Chu, 1978; Hamperl, 1962a). In all cases, the ultrastructural features of oncocytes are characteristic and consist of numerous, tightly-packed, enlarged, atypical mitochondria completely filling the cytoplasm of the cell. This report docu- ments, by electron microscopy and histochemistry, the presence of oncocytes resembling cytomegalic inclusion disease in the salivary glands of Afiffcan hedgehogs and speculates on the origin of these oncocytes.

Mater ia l s and M e t h o d s

One adult male and two adult female hedgehogs, caught in the wild, died shortly after arrival from Africa. A complete necropsy was performed on all animals. Tissue samples were fixed in 10 per cent buffered formalin, processed routinely, stained with haematoxylin and eosin (HE), phosphotungstic acid haematoxylin (PTAH), lead haematoxylin, acid-fast, feulgen, Luxol fast blue, sulphuric acid-permanganate aldehyde-fuchsin, Feyrter's thionine inclusion stain (Feyrter, 1947), Vogt's cresyl violet and periodic acid-Schiff (PAS).

Formalin-fixed submandibular salivary gland was prepared for electron microscopy by post-fixation in 1 per cent osmium tetrooxide, dehydrated with graded alcohols and embedded in epoxy resin. Fields for electron microscopy were selected from 1 gm sections stained with 1 per cent toluidine blue. Sections 60 to 90 nm in thickness were stained with lead citrate and uranyl acetate and viewed with a JEOL CX100 transmission electron microscope.

R e s u l t s

Upon arrival from Africa, all three hedgehogs were in extremely poor condition. Two of the three had blood-stained diarrhoea and were positive fbr strongy- loides ova on faecal flotation, but adult nematodes were not identified on necropsy. All animals were markedly dehydrated and emaciated. The skin on the tail, perineum and ventral aspects of the hind feet was excoriated and had numerous fleas and ticks (Amblyomma nultali). There were minimal subcutaneous and abdominal fat stores. The stomach contained varying numbers (few to numerous) of spirurid-type nematodes. The cause of death was related to parasitism, emaciation and dehydration in all three animals.

Salivary Gland Oncocytes 85

The submandibular salivary glands contained multiple foci of markedly cytomegalic cells in all animals. Occasional foci had a mild to moderate infiltrate of lymphocytes at the periphery (Fig. 1). Cytomegalic cells were present in the minor salivary glands in only one of the females, and the parotid salivary gland in the male. The parotid gland in the male hedgehog contained a lymphocytic infiltrate similar to that of the submandibular salivary glands. The remaining minor, parotid and sublingual salivary glands were unremark- able. In all three animals, no cytomegalic cells were present in other organs examined, including the thyroid, adrenal, pancreas and lacrimal glands (only one animal examined). The parathyroid could not be identified in any of the hedgehogs, probably owing to its extremely small size.

The majority of cytomegalic cells appeared to occur in the glandular acini and, infrequently, in ductules. The cells were 35 to 4.0 I~m in diameter with well delineated cell membranes, abundant acidophilic finely granular to hyalin cytoplasm, enlarged nuclei with marginalized clumped chromatin and a central eosinophilic nucleolus 6 to 8 t~m in diameter (Fig. 2). Occasional cells contained large, multiple clear cytoplasmic vacuoles. The remaining pyrami- dal acinar cells and ducts were unremarkable.

Histochemical staining of the cytomegalic cells revealed varying amounts of metachromic cytoplasmic granules with Feyrter's thionine inclusion stain. Scattered cells at the periphery of the cytomegalic foci contained PAS-positive cytoplasmic granules. The cytoplasm was positive for phospholipids with Luxol fast blue, but did not have a strong affinity for the haematoxylin of PTAH (Allen and Thorburn, 1981) or cresyl violet (Hamperl, 1962b) as previously reported. The cytomegalic cell nucleus was negative for heavy metals with feulgen, acid-fast, Luxol fast blue, lead haematoxylin or sulphuric acid-permanganate aldehyde-fuchsin stains.

Ultrastructurally, the cytomegalic cells occurred in well-delineated foci separated from pyramidal acinar cells (Fig. 3). Cytomegalic cells were attached to adjacent cells by cell junctions and desmosomes. The cytoplasm of the cells contained numerous, tightly-packed, often bizarre enlarged mito- chondria that sometimes indented one another (Fig. 4:). The majority of mitochondria had a normal structure, but were 2 to 3 times normal size, while others had striking alterations consisting of increased number and length of cristae with altered orientation. Cristae were disposed longitudinally in close array and, occasionally, in concentric rings. In the remaining cytoplasm, a small amount of rough endoplasmic reticulum and occasional glycogen granules limited by a single membrane were present.

The nuclei of the cytomegalic cells were round to oval with clumped marginalized chromatin. The remaining chromatin was centrally located and consisted of a dense central core often associated with the nucleolus (Fig. 5). Nuclear or cytoplasmic virions were not present.

At the periphery of the foci of cytomegalic cells, there were "transitional" cells which contained markedly fewer enlarged mJtochrondria, numerous golgi, abundant rough endoplasmic reticulum and membrane-bound glycogen granules that were similar to previous reports (McGregor el al., 1978). Pyramidal acinar cells were unremarkable.

86 S.R. Brunnert e t al .

Fig. 1.

Fig. 2.

Submandibular saliwtry gland, AIi'ican hedgehog. Multiple loci ofcytomegalic cells with a mild to moderate infiltrate of lymphocytes at the periphery. HE. Bar= 100 p.m.

Submandibular salivary gland, Afi'ican hedgehog. Focus of cytomegalic cells with abundant eosinophilic granular to hyalin cytoplasm, marginated and clumped nuclear ehromatin (arrows), and prominent central nucleolus (arrow heads). HE. Bar= 60 p.m.

S a l i v a r y G l a n d O n c o c y t e s 87

Fig. 3.

Fig. 4..

Electron micrograph. Focus of oncocytes with abundant cytoplasmic mitochondria adjacent to pyramidal acinar cells that contain numerous cytoplasmic secretory granules. Uranyl acetate and lead citrate. Bar=3 btm.

Electron micrograph. Oneocyte with numerous, tlghtly-packed, enlarged mitochondria. Numerous desmosomes (arrow heads) are present between oncocytes. Uranyl acetate and lead citrate. Bar = 1 am.

8 8 S . R . B r u n n e r t e t al .

Fig. 5. Electron micrograph. Oncocyte nucleus with clumped marginated chromatln with the remaining chromatin consisting of a dense central core associated with the nucleolus (N). Uranyl acetate and lead citrate. Bar= 6 Jam.

Discussion

The ultrastructural and histochemical findings of the salivary gland cyto- megalic cells in the African hedgehogs were consistent with oncocytes. The characteristic features of oncocytes (oxyphil cells) are cytomegaly with well demarcated cell membranes, brightly eosinophilic granular or hyalin cyto- plasm, metachromasia with Feyrter's inclusion stain using thionine or cresyl violet and cytoplasmic lipoproteins demonstrated with Luxol fast blue (Hamperl, 1962a and b; Tremblay, 1969). Ultrastructurally, oncocytes con- tain numerous, tightly-packed, enlarged, atypical mitochondria in the cyto- plasm (Tandler, 1966; McGregor et al., 1978).

Classic morphological descriptions of the oncocyte nucleus vary from small hyperchromatic oval nuclei to large pleomorphic vesicular nuclei with margi- nalized chromatin and a single distinct nucleolus (Hamperl, 1962b). In this report, the oncocyte nucleus was consistent with the latter description and had the appearance of an inclusion body on light microscopy.

Oncocytes originate from various epithelial cells, but lose their respective organ-specific special features in favour of a novel differentiation (Hamperl, 1962b). In the salivary glands, oncocytes occur with increased frequency with aging in man (Tandler, 1966), rats (Bogart, 1970; Andrew, 1949), mice (Andrew, 1956), canines (Hamperl, 1962b) and rabbits (Hamperl, 1962b). Other reports describe cytomegalic cells which may represent oncocytes in the salivary glands of aging Syrian hamsters (Schmidt, Eason, Hubbard, Young and Eisenbrandt, 1983).

Cytomegalovirus infection has been documented in a variety of animal species including insectivores (Hanshaw, 1968; Mineda, 1981). Hedgehogs

Salivary Gland Oncocytes 89

have also been reported to be serologically positive against herpes antigen by a complement-fixation test (Sixl, K6ck, Withalm and Stunzner, 1989). Cytome- galovirus-infected cells usually occur singly or in small dissociated clusters. In contrast, oncocytes occur in small to large well-delineated clusters with tight cellular junctions, as seen in this report. This morphological feature can be used along with histochemical stains to differentiate oncocytes from cytomega- lovirus by light microscopy.

Heavy metal toxicity can induce intranuclear inclusion bodies, especially in renal tubular epithelial cells (Landing and Nakai, 1959; Richter, Kress and Cornwall, 1968). The insectivore may be particularly susceptible to heavy metal toxicity since these animals are predators of soil macro-invertebrates that can be heavy metal bio-accumulators (Ma, 1987). Heavy metal inclusion bodies stain positive with feulgen, acid-fast, Luxol fast blue, lead haematoxylin and sulphuric acid-permanganate aldehyde-fuchsin stains (Landing and Nakai, 1959). Ultrastructurally, the inclusion consists of a dense central chromatin core with an outer fibrillary zone (Richter et al., 1968). In this study, the oncocyte nuclei were negative with the above listed stains and the nuclear ultrastructural findings were not consistent with heavy metal toxicity. This suggests that heavy metal toxicity is not related to the formation of oncocytes in the hedgehog.

Intranuclear inclusion bodies, seen by light microscopic examination, may also be as a result of cytoplasmic invaginations (van Zwieten and Hollander, 1985; Gaertner, Lindsey and Stevens, 1988) or age-related clumping of the nucleolar-associated and peripheral chromatin (Bogart, 1970). In this report, the latter is the most likely cause of the prominent nucleoli giving the impression of inclusion bodies.

In the African hedgehog, the presence of lymphocytes surrounding the loci of oncocytes might suggest an autoimmune etiology similar to Sj6gren's syndrome seen in man and animals (Daniels, 1986). However, focal lympho- cytic infiltrates of salivary glands are not limited to Sj6gren's syndrome, but may be seen in a variety of chronic systemic inflammatory diseases (Lindahl and Hedfors, 1986). In this case, the pathogenesis of the lymphocytic infiltrate is unknown.

This study demonstrates, by histochemical and ultrastructural methods, the presence of oncocytes in the submandibular salivary gland of three African hedgehogs. Previous reports had mistaken oncocytes for a possible cytomegalo- virus infection in Syrian hamsters (Schmidt et al., 1983) and insectivores (Karstad, 1975; Cosgrove, 1986), since the cytomegalic cells were limited to the salivary glands. The presence of oncocytes in the salivary gland of the African hedgehog may represent a normal aging change as seen in other animal species. Since over 50 per cent of the salivary glands from African hedgehogs in this study and in those animals examined by Karstad contain oncocytes, they may represent a good animal model to study oncocyte pathogenesis and function.

Acknowledgments

This work was partially supported by NIH grant 1P40 RR 040326-02. We thank Ms Susan Decker for technical assistance with the electron microscopy and Dr M. Yoshioka for his help with the language translation of a rei~rence.

90 S.R. Brunnert e t al.

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I Received, September 19th, 1990"] Accepted, February 14th, 1991 ..]