Papillomatosis associated with papillomavirus infection has been described in ... serum for 5 min at 378C, before applying rabbit anti-bovine papillomavirus ...
Veterinary Research Communications, 24 (2000) 197^202 # 2000 Kluwer Academic Publishers. Printed in the Netherlands
An Apparent Outbreak of Cutaneous Papillomatosis in Merino Sheep in Patagonia, Argentina F.A. Uzal1*, A. Latorraca2, M. Ghoddusi1, M. Horn3, M. Adamson2, W.R. Kelly1 and R. Schenkel2 1 School of Veterinary Sciences and Animal Production, The University of Queensland, Brisbane, Queensland, Australia; 2Instituto Nacional de Tecnolog|¨ a Agropecuaria (INTA), Trelew, Argentina; 3Immunohistology Laboratory, Princess Alexandra Hospital, Ipswich Road, Woolloongabba, Queensland, Australia *Correspondence: Instituto Nacional de Tecnolog|¨ a Agropecuaria (INTA), CC 277 (8400) Bariloche, Argentina Uzal, F.A., Latorraca, A., Ghoddusi, M., Horn, M., Adamson, M., Kelly, W.R. and Schenkel, R., 2000. An apparent outbreak of cutaneous papillomatosis in Merino sheep in Patagonia, Argentina. Veterinary Research Communications, 24(3), 197^202 ABSTRACT A retrospective study was performed on skin samples from an outbreak of cutaneous papillomatosis in Merino sheep that occurred in 1995. The samples were processed for routine histology, electron microscopy and immunocytochemistry for papilloma viruses. Particles of approximately 55 nm diameter were found in some nuclei of the stratum granulosum cells, while immunocytochemistry gave positive staining of cell nuclei in this layer. This study con¢rms that papillomas associated with papillomaviruses occur in sheep in Patagonia. Keywords: diagnosis, electron microscopy, histology, immunocytochemistry, papillomavirus, sheep Abbreviations: DAB, diaminobenzidine tetrahydrochloride; PBS, phosphate-bu¡ered saline, pH 7.4
INTRODUCTION Papillomatosis associated with papillomavirus infection has been described in many animal species and in humans (Moulton, 1954; Sundberg, 1987; Zur Hausen, 1996). However, there are few reports of this condition in sheep and, in this species, papillomas have usually been identi¢ed by morphology alone (Head, 1965; James et al., 1967). Gibbs and colleagues (1975) reported papillomas on the foreleg of Southdown cross sheep in England and demonstrated particles that were ultrastructurally similar in size and symmetry to papillomaviruses in these lesions. They were able to transmit the lesion to other sheep, using cell-free and bacteria-free inocula prepared from the ovine warts. Papillomavirus-like virions were detected by negative stain electron microscopy in hyperkeratotic scales on the perineum of sheep (Vanselow and Spradbrow, 1983) and in papillomatous areas on the face and in highly keratinized horn on the ear of a Merino sheep in Australia (Vanselow and Spradbrow, 1982). 197
198
This is a report of retrospective identi¢cation of papillomavirus in samples from an outbreak of cutaneous papillomatosis in Merino sheep that occurred in 1995 in southern Argentina. MATERIALS AND METHODS The animals belonged to an extensive sheep farm in Chubut province, Patagonia (approximately 458S). The climate is cold semidesert (average annual precipitation of 161 mm). Sheep are raised on natural pastures. All a¡ected animals were Merinos about 19 months old. They were mustered in December 1994 for shearing, when a 10-mm-long lesion was observed on only one ram in a group of 63 animals. No lesions were observed in another group of 403 ewes on the same property that were examined at the same time. Both £ocks were examined again 3 months later, when the lesion previously observed on the ram had grown to a length of approximately 30 mm. Also at this time, similar lesions were observed in 20 (5%) of the 403 ewes. These lesions were papillary growths, 5^40 mm long, with a base of 3^5 mm. They were also found on areas lacking wool, mostly on the skin of the muzzle and on the front and rear legs, where they were located on the dorsal and lateral aspects, usually between the coronary band and fetlock joint. No other clinical alterations were observed in the a¡ected animals. The animals were re-examined 9 months later and no skin lesions were observed. The rams and ewes had not been in contact between the ¢rst and the second examination, although they had been in contact during the previous mating season, approximately 6 months before the ¢rst examination. In March 1995, skin biopsies were obtained from ¢ve animals and ¢xed in 10% bu¡ered formalin. Para¤n sections were cut and stained with haematoxylin and eosin using routine methods. Tissue samples that had been stored in formalin for approximately 3 years were trimmed to 1-mm3 pieces and post-¢xed overnight in glutaraldehyde before being processed for transmission electron microscopy. The tissues were post-¢xed in potassium ferrocyanide^osmium ¢xative for 1 h, dehydrated in ascending concentrations of acetone and embedded in medium Spurr's resin (ProSciTech Co., Brisbane, Australia). Survey sections of 1 mm thickness were prepared and stained with toluidine blue. Ultrathin sections from selected areas were cut, placed on formvar-coated copper grids, contrasted with lead citrate for 2 min followed by uranyl acetate for 5 min, and examined in a transmission electron microscope (Jeol, Tokyo, Japan). It was decided to perform immunocytochemistry using an antibody against papillomavirus, and, because the original para¤n block was not available, a new block was made from tissues that had been kept in formalin for 3 years. Sections were cut at 3 mm, dewaxed and rehydrated. Endogenous peroxidase was blocked with 3% H2O2 at room temperature for 5 min. Non-speci¢c staining was blocked with 5% normal goat serum for 5 min at 378C, before applying rabbit anti-bovine papillomavirus primary antibody (Dako, Copenhagen, Denmark) for 15 min at 378C. After washing in phosphate-bu¡ered saline, pH 7.4 (PBS), biotinylated goat anti-rabbit second antibody (Vector, Beverly, MA, USA) was applied for 15 min at 378C. After washing in PBS,
199
peroxidase-labelled streptavidin (Jackson, West Grove, PN, USA) was applied for 10 min at 378C prior to another washing cycle. The sections were incubated in diaminobenzidine tetrahydrochloride (DAB) for approximately 5 min, washed in water and counterstained with Mayer's haematoxylin. RESULTS Histologically, the lesions consisted of long, thin dermal papillae covered by a markedly hyperplastic epithelium (Figure 1), over which there was a thick keratinous layer in which there was nuclear retention. In the stratum granulosum, a moderate number of cells had their nuclei surrounded by a ring of clear cytoplasm (Figure 1). Also in this layer, a few cells were observed with clear vacuoles adjacent to one or both poles of the nuclei. Cytoplasmic eosinophilic inclusions were visible in some cells in this layer. The dermis was thickened by dense connective tissue, with irregular plump cells with loose nuclear chromatin.
Figure 1. Stratum corneum and stratum granulosum of a sheep papilloma. Notice the hyperkeratosis and the presence of cells with clear cytoplasm around the nucleus (open arrow) and of cells with vesicles on both poles of the nucleus (arrow). HE, 6400
200
At the electron-microscopic level, the most striking ¢nding was the presence of numerous roughly spherical particles in some of the nuclei of the stratum granulosum. These particles were approximately 55 nm in diameter (Figure 2), and their size and shape were consistent with previous descriptions of papillomaviruses (P¢ster and Fuchs, 1994), which were de¢ned as having icosohedral capsids, 55 nm in diameter.
Figure 2. Electron micrograph of papillomavirus-like particles in the nucleus of a cell in the stratum granulosum of a sheep papilloma. The arrows show the nuclear membrane; 627 000. Insert: higher magni¢cation of the viral particles; 654 000
Immunohistochemistry showed strong positive staining of approximately 25% of the cell nuclei in the stratum granulosum (Figure 3). No staining was observed in sections from the same block processed using normal rabbit serum, or in negative control sections processed with rabbit anti-bovine papillomavirus or normal rabbit serum. DISCUSSION This study con¢rmed that sheep papillomas associated with papillomaviruses occur in Patagonia. The diagnosis of viral papillomatosis was based on the clinical ¢ndings, on the histological appearance of the lesions, on the presence of papillomavirus-like particles in the nuclei of the cells in the stratum granulosum, and on the positive staining of some
201
Figure 3. Positive reaction to an anti-bovine papillomavirus antibody in the nuclei of the stratum granulosum cells in sheep papilloma. Avidin-streptavidin, 6320
of these nuclei with the immunocytochemical technique for bovine papilloma virus. Epidemiological information about papillomas in sheep is practically non-existent. The presence of these lesions on the front and lateral aspects of the legs and on the muzzle, both areas that are most susceptible to minor trauma during grazing, suggests that this played a role in the transmission of the virus. This mechanism of transmission has been proposed for the transmission of contagious pustular dermatitis (orf) in sheep (Gardiner et al., 1967). Two types of cutaneous ovine papilloma viruses (OvPV-1 and -2) were recently isolated at the Infectious Diseases Laboratories, Institute of Medical and Veterinary Services, Adelaide, Australia (personal communication by journal referee), and their genomes were cloned and sequenced. It would be of epidemiological interest to establish whether the papillomaviruses found in our study belong to either of these types. Further studies will be necessary to determine the type of papillomaviruses in sheep in Patagonia. Papillomaviruses have been associated with a number of hyperplastic and neoplastic lesions in a wide variety of animal species (Sundberg, 1987). In addition, papillomas are believed to be precursor lesions for squamous cell carcinomas in goats (Moulton, 1954), cattle (Campo, 1997) and humans (Zur Hausen, 1996). Vanselow and Spradbrow (1982) described papillomas, corni¢ed horns and squamous cell carcinomas on the wool-free areas of the faces of Merino sheep, and suggested that ovine facial and aural squamous cell carcinomas have a complex aetiology involving unpigmented skin, exposure to sunlight and infection with papillomavirus. Squamous cell carcinoma of
202
the ear has been described in Merino sheep in Patagonia (Olaechea et al., 1991), although we are not aware of its occurrence on the farm from which this study originated. An anti-bovine papillomavirus antibody was used in this study because an antiovine papillomavirus antibody was not available. Nevertheless, a strong signal was obtained with the anti-bovine virus reagent. This was all the more remarkable because the material had been stored for so long in formalin, which might well have su¤ciently denatured the relevant antigenic determinants as to have precluded speci¢c binding (Pettigrew, 1989). ACKNOWLEDGEMENTS This study was funded by the University of Queensland, Australia, and by The National Institute of Agricultural Technology and the National Council of Scienti¢c and Technical Research of Argentina. REFERENCES Campo, M.S., 1997. Bovine papillomavirus and cancer. Veterinary Journal, 154, 175^188 Gardiner, M.R., Craig, J. and Nairn, M.E., 1967. An unusual outbreak of contagious ecthyma (scabby mouth) in sheep. Australian Veterinary Journal, 43, 163^165 Gibbs, E.P.J., Smale, C.J. and Lawman, M.J.P., 1975. Warts in sheep. Journal of Comparative Pathology, 85, 327^334 Head, K.W., 1965. Some data concerning the distribution of skin tumours in animals. In: A.J. Rooks and G.S. Walton (eds), Comparative Physiology and Pathology of the Skin, (Davis and Co., Philadelphia), 615^636 James, C.K., Ramo Rao P. and Sastry, G.A., 1967. Report on ¢ve ovine neoplasms. Ceylon Veterinary Journal, 15, 138 Moulton, J.E., 1954. Cutaneous papillomas on the udder of milk goats. North American Veterinarian, 35, 29^33 Olaechea, F.V., Uzal, F.A., Robles, C.A., Arrigo, J.L. and Latorraca, A., 1991. Carcinoma de celulas escamosas en ovinos de Patagonia. Proceedings of the 7th Annual Meeting of the Argentine Association of Laboratory Veterinary Diagnosticians, Buenos Aires, Argentina, 1991, pp. 64 Pettigrew, N.M., 1989. Techniques in immunocytochemistry. Application to diagnostic pathology. Archives of Pathology and Laboratory Medicine, 113, 641^644 P¢ster, H. and Fuchs, P.G., 1994. Anatomy, taxonomy and evolution of papillomaviruses. Intervirology, 37, 143^149 Sundberg, J.P., 1987. Papillomavirus infections in animals. In: K. Syrjanen, L., Gissmann and L.G. Koss (eds), Papilloma-viruses and Human Diseases, (Springer-Verlag, Berlin), 40^103 Vanselow, B.A. and Spradbrow, P.B., 1982. Papillomaviruses, papillomas and squamous cell carcinomas in sheep. Veterinary Record, 110, 561^562 Vanselow, B.A. and Spradbrow, P.B., 1983. Squamous cell carcinomas of the vulva, hyperkeratosis and papillomaviruses in a ewe. Australian Veterinary Journal, 60, 194^195 Zur Hausen, H., 1996. Papillomavirus infections ^ a major cause of human cancers. Biochimica et Biophysica Acta, 1288, F55^78 (Accepted: 30 August 1999)
