J Vet Diagn Invest 3:36-38 (1991)
Genital bovine papillomavirus infection in Saudi Arabia El Tayb Ebu Elzein, John P. Sundberg, Fadhel M. Housawi, Ahmed A. Gameel, Ramadan O. Ramadan, Magdi M. Hassanein Abstract. Genital bovine papillomavirus infection was observed for the first time in the Al-Ahsa region of Saudi Arabia. The disease involved 1 female and 2 male 2-4-year-old crossbred cattle. Fibropapillomas (warts) were limited to the prepuce and vulva. Electron micrographs of thin sections of the lesions revealed the presence of intranuclear viruslike particles. Using a broadly cross-reactive rabbit polyclonal antiserum directed against papillomavirus group-specific antigens, the infection was confirmed by immunohistochemical staining of paraffin-embedded tissues to be due to a papillomavirus. Staining with a series of monoclonal antibodies of various specificities indicated that the virus was bovine papillomavirus type 1. Attempts to propagate the virus by inoculation of tumor homogenates onto chorioallantoic membranes of chicken embryos were unsuccessful.
most definitive methods of typing the papillomaviruses. In vitro culture methods have not been successful for propagation of this genus of virus.7 The purpose of this paper is to describe the first case of genital bovine papillomavirus infection in domestic cattle in the Kingdom of Saudi Arabia and the typing of virus using a panel of MAbs when frozen tissue is not available.
Bovine papillomaviruses (BPVs) are viruses that belong to the genus Papillomavirus of the family Papovaviridae. These viruses are nonenveloped, have an icosahedral capsomeric pattern, and contain a doublestranded covalently closed circular DNA molecule.5 The BPVs are readily distinguished by characteristic restriction endonuclease cleavage patterns of their genomes and the degree of polynucleotide sequence homology. 2,3 At least 6 BPVs have been identified. Types 1, 2, and 5 induce fibropapillomas of the skin or teat and have nucleic acids of very similar molecular weight and a high degree of homology. A second group, types 3, 4, and 6, induce true papillomas of the esophagus or teat and are molecularly similar to each other but different from types 1, 2, and 5. Therefore, 2 types (A and B) have been proposed, with each type consisting of at least 3 subtypes (for review, see references 7 and 8). Various immunohistochemical techniques utilizing a papillomavirus group-specific antiserum have been used successfully to identify papillomavirus antigens in a variety of mammalian and avian neoplasms.6,9 The antiserum technique is a rapid and reliable screening method. The late genes of the BPV-1 genome code for structural proteins with over 12 epitopes.4 Monoclonal antibodies (MAbs) directed against many of these epitopes are useful for partially typing papillomaviruses by immunohistochemistry.4 Restriction endonuclease digestion combined with high-stringency Southern blot analysis are still considered to be the
Materials and methods Clinical investigation. Three crossbred cattle, 2 males and 1 female 2-4 years of age, were examined at the King Faisal University Veterinary Teaching Hospital at Al-Ahsa. The animals were healthy on physical examination except for multiple firm raised gray to black verrucous tumors affecting the vulva and prepuce. The tumors ranged in size from a few millimeters (barely perceptible) to 5-7 cm in diameter. The larger tumors were ulcerated. Histopathology. Tumors were surgically removed, fixed in neutral buffered 10% formalin, processed routinely, serially sectioned at 5 µm, and stained with hematoxylin and eosin, periodic acid-Schiff, Masson’s trichrome, and Van Gieson’s stains. Inoculation of chorioallantoic membranes. Portions of the tumors collected for histopathology were stored in 50% glycerol in phosphate-buffered saline (PBS), pH 7.4. The epithelial portion of the tumor was removed with scalpels, finely diced, and homogenized in PBS with sand in a mortar and pestle. The debris was removed by low-speed centrifugation (3,000 x g). Penicillin (1,000 IU/ml), gentamycin (200 pg/ ml), and mycostatin (50 U/ml) were added to the supernatant, which was then inoculated into 11-day-old chicken embryos via the chorioallantoic membrane (CAM) as described.’ Eggs were examined daily. Dead embryos were discarded within the first 48 hr. Five egg passages were carried out. Electron microscopy. Freshly removed tumors and CAMS from inoculated chicken embryos were finely diced (< 1 mm
From the College of Veterinary Medicine, King Faisal University, Hoffuf, PO Box 1757, Saudi Arabia (Elzein, Housawi, Gameel, Ramadan, Hassanien), and The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609-0800 (Sundberg). Received for publication June 1, 1990. 36
Downloaded from vdi.sagepub.com at NEW YORK UNIV LIBRARY on April 24, 2015
Genital bovine papillomatosis
37
Figure 1. Section of a skin tumor from a cow. Bovine papillomavirus type 1 antigens are demonstrated by mouse monoclonal antibody AU-2 as brown-staining nuclei (arrowhead) within cells of the stratum granulosum of the epidermis. Immunoperoxidase stain, light green counterstain, 400 x .
thick) and immediately fixed in cacodylate-buffered 2% glutaraldehyde for 3 hr and postfixed in 1% osmium tetroxide.
Tissues were processed and sectioned routinely, stained with uranyl acetate and Reynold’s lead citrate, and examined in an electron microscope operated at 60 kV. Immunohistochemistry. Serial sections of the tumors were deparaffinized through a series of xylenes and graded ethanols. Nonspecific protein binding was blocked by incubation in 10% ovalbumin.9 A rabbit polyclonal antibody directed against papillomavirus group-specific antigensa was used as the primary antiserum. Affinity-purified rabbit IgGa was used as a negative control serum. A series of seven mouse MAbs 4 with various specificities (AU-l, -2, -3, -4, -5, -6, and 1H8) and normal mouse serum (negative control) were also used. The avidin-biotin complex (ABC) techniqueb was used to detect the bound antiserum, and diaminobenzidinec was used as the chromogen. Sections were counterstained with light green. Sections of bovine cutaneous fibropapillomas, confirmed by restriction analysis to contain either BPV-1 or BPV-2, were used as positive controls.
Results Histopathology. Microscopic features of all 3 tumors were identical. There was marked proliferation of the epithelium as well as proliferation of dermal fibroblasts, giving the tumors a broad papillary pattern. All layers of the epidermis were markedly thickened. Prominent changes were observed within the stratum granulosum, where individual and clusters of cells had abundant clear cytoplasm, often containing large pleo-
morphic keratohyalinlike granules. These cellular changes are typical of what have been called koilocytes, clear cells, or pale cells, changes considered to be typical for productive papillomavirus infections.7 The dermis consisted of densely packed fibroblasts and dense irregular collagenous connective tissue. The morphologic patterns were typical of fibropapillomas of papillomavirus etiology. Immunohistochemistry. Cells within the stratum granulosum of the proliferating epidermis that were undergoing degenerative changes resembling koilocytosis contained dark brown-staining nuclei (Fig. 1) typical of positive reactions for papillomavirus antigens. The nuclear staining continued into the stratum corneum; however, the nuclear size increased, then became poorly defined as would be expected in this layer. Positive nuclear staining was detected in all serial sections stained with both the monoclonal and polyclonal antibodies. The AU-2 antibody, which distinguishes BPV-1 from BPV-2 at high dilutions, stained virioncontaining cells at dilutions of 1:2,000. The BPV-2 control sections were stained at 1:100 dilutions but not at higher dilutions. This verified that the papillomavirus in these tissues was BPV-1. Chorioallantoic membrane inoculation. Conspicuous turbidity and thickening were observed on the CAM S of the chicken embryos 4-5 days after inoculation with the tumor suspensions on all CAM passages. No discrete lesions were detected on the CAM S
Downloaded from vdi.sagepub.com at NEW YORK UNIV LIBRARY on April 24, 2015
38
Elzein et al.
at any passage. Thin-section electron microscopy of infected CAMS did not reveal the presence of viruslike particles. Thin-section electron microscopy. Viruslike particles, 30-35 nm in diameter, were observed within nuclei of cells in the stratum granulosum in multiple sections taken from tumors of all 3 cattle. The particles formed large crystalline arrays.
been utilized effectively for prevention but not for treatment. Artificial insemination has virtually eliminated this disease in North America in herds using this breeding system. Acknowledgements This work was supported in part by grant number 2174 from the Council for Tobacco Research (JPS). We thank Dr. A. B. Jenson for graciously providing the monoclonal antibodies used in this study, Ann Higgins for her technical expertise in immunohistochemistry, and Mr. P. Prentis for the electron microscopy.
Discussion This paper describes the first case of genital bovine fibropapillomatosis in the Kingdom of Saudi Arabia and the Gulf Region and provides evidence that the Sources and manufacturers lesions were caused by a papillomavirus. Methods for the detection and prevention of this disease are needed a. DAKP Corp., Santa Barbara, CA. b. Vector Chemicals, Burlingame, CA. to prevent widespread dissemination as Saudi Arabia c. Sigma Chemical Corp., St. Louis, MO. begins to expand its cattle production. References The etiologic agent of genital fibropapillomas in cattle is generally considered to be BPV-1 or -2. 7 Molec- 1. Bruner DW, Gillespie JH: 1973, Hagan’s infectious diseases of ular studies are usually required to make a definitive domestic animals, 6th ed., pp. 1311-1314. Cornell University diagnosis. Immunohistochemistry and electron miPress, Ithaca, NY. croscopy are 2 generally available techniques for con- 2. Coggin JR, zur Hausen H: 1979, Workshop on papillomaviruses and cancer. Cancer Res 39:545-546. firming a papillomavirus etiology but are not adequate 3. Lancaster WD, Olson C: 1978, Demonstration of two distinct for typing. Panels of MAbs directed against various classes of bovine papilloma virus. Virology 89:371-379. BPV-1 epitopes can now be used to type or semitype 4. Lim P, Jenson AB, Cowsert L, et al.: 1990, Distribution and specific identification of papillomavirus major capsid protein epivarious papillomaviruses in tissue sections4 and were topes by immunocytochemistry and epitope scanning of synthetic useful in the cases described here for confirming a peptides. J Infect Dis 162: 1263-1269. BPV-1 etiology. The most accepted method of papil5. Pfister H: 1987, Papillomaviruses. General description, taxonlomavirus typing remains viral (low molecular weight) omy, and classification. In: The Papovaviridae, ed. Salzman NP, DNA extraction, restriction endonuclease digestion, Howley PM, vol. 2, The papillomaviruses, pp. l-38. Plenum Press, New York, NY. and high-stringency Southern blot analysis with appropriate controls. However, frozen tissues are re- 6. Sundberg JP: 1987, Animal models for papillomavirus research. Contrib Oncol 24: 11-38. quired for this approach. Immunohistochemical tech7. Sundberg JP: 1987, Papillomavirus infections in animals. In: niques provide an alternative when frozen tissues are Papillomaviruses and human diseases, ed. Syrjanen K, Gissmann not available or when shipping conditions preclude L, Koss LG, pp. 40-103. Springer-Verlag, Heidelberg. proper handling of such specimens. Information on 8. Sundberg JP: 1990, Bovine papillomaviruses. In: Diagnostic veterinary virology: a practitioner’s guide, ed. Castro AE, Heuschele papillomavirus type can then be utilized to develop WP (in press). Williams and Wilkins, Baltimore, MD. methods of prevention or treatment. 9. Sundberg JP, Junge RE, Lancaster WD: 1984, ImmunoperoxiMany approaches are available for the prevention dase localization of papillomaviruses in hyperplastic and neoof bovine genital fibropapillomatosis. The best method plastic epithelial lesions of animals. Am J Vet Res 45: 1441-1446. is to maintain a clean herd.8 Autogenous vaccines have
Downloaded from vdi.sagepub.com at NEW YORK UNIV LIBRARY on April 24, 2015
Genital bovine papillomavirus infection in Saudi Arabia El Tayb Ebu Elzein, John P. Sundberg, Fadhel M. Housawi, Ahmed A. Gameel, Ramadan O. Ramadan, Magdi M. Hassanein Abstract. Genital bovine papillomavirus infection was observed for the first time in the Al-Ahsa region of Saudi Arabia. The disease involved 1 female and 2 male 2-4-year-old crossbred cattle. Fibropapillomas (warts) were limited to the prepuce and vulva. Electron micrographs of thin sections of the lesions revealed the presence of intranuclear viruslike particles. Using a broadly cross-reactive rabbit polyclonal antiserum directed against papillomavirus group-specific antigens, the infection was confirmed by immunohistochemical staining of paraffin-embedded tissues to be due to a papillomavirus. Staining with a series of monoclonal antibodies of various specificities indicated that the virus was bovine papillomavirus type 1. Attempts to propagate the virus by inoculation of tumor homogenates onto chorioallantoic membranes of chicken embryos were unsuccessful.
most definitive methods of typing the papillomaviruses. In vitro culture methods have not been successful for propagation of this genus of virus.7 The purpose of this paper is to describe the first case of genital bovine papillomavirus infection in domestic cattle in the Kingdom of Saudi Arabia and the typing of virus using a panel of MAbs when frozen tissue is not available.
Bovine papillomaviruses (BPVs) are viruses that belong to the genus Papillomavirus of the family Papovaviridae. These viruses are nonenveloped, have an icosahedral capsomeric pattern, and contain a doublestranded covalently closed circular DNA molecule.5 The BPVs are readily distinguished by characteristic restriction endonuclease cleavage patterns of their genomes and the degree of polynucleotide sequence homology. 2,3 At least 6 BPVs have been identified. Types 1, 2, and 5 induce fibropapillomas of the skin or teat and have nucleic acids of very similar molecular weight and a high degree of homology. A second group, types 3, 4, and 6, induce true papillomas of the esophagus or teat and are molecularly similar to each other but different from types 1, 2, and 5. Therefore, 2 types (A and B) have been proposed, with each type consisting of at least 3 subtypes (for review, see references 7 and 8). Various immunohistochemical techniques utilizing a papillomavirus group-specific antiserum have been used successfully to identify papillomavirus antigens in a variety of mammalian and avian neoplasms.6,9 The antiserum technique is a rapid and reliable screening method. The late genes of the BPV-1 genome code for structural proteins with over 12 epitopes.4 Monoclonal antibodies (MAbs) directed against many of these epitopes are useful for partially typing papillomaviruses by immunohistochemistry.4 Restriction endonuclease digestion combined with high-stringency Southern blot analysis are still considered to be the
Materials and methods Clinical investigation. Three crossbred cattle, 2 males and 1 female 2-4 years of age, were examined at the King Faisal University Veterinary Teaching Hospital at Al-Ahsa. The animals were healthy on physical examination except for multiple firm raised gray to black verrucous tumors affecting the vulva and prepuce. The tumors ranged in size from a few millimeters (barely perceptible) to 5-7 cm in diameter. The larger tumors were ulcerated. Histopathology. Tumors were surgically removed, fixed in neutral buffered 10% formalin, processed routinely, serially sectioned at 5 µm, and stained with hematoxylin and eosin, periodic acid-Schiff, Masson’s trichrome, and Van Gieson’s stains. Inoculation of chorioallantoic membranes. Portions of the tumors collected for histopathology were stored in 50% glycerol in phosphate-buffered saline (PBS), pH 7.4. The epithelial portion of the tumor was removed with scalpels, finely diced, and homogenized in PBS with sand in a mortar and pestle. The debris was removed by low-speed centrifugation (3,000 x g). Penicillin (1,000 IU/ml), gentamycin (200 pg/ ml), and mycostatin (50 U/ml) were added to the supernatant, which was then inoculated into 11-day-old chicken embryos via the chorioallantoic membrane (CAM) as described.’ Eggs were examined daily. Dead embryos were discarded within the first 48 hr. Five egg passages were carried out. Electron microscopy. Freshly removed tumors and CAMS from inoculated chicken embryos were finely diced (< 1 mm
From the College of Veterinary Medicine, King Faisal University, Hoffuf, PO Box 1757, Saudi Arabia (Elzein, Housawi, Gameel, Ramadan, Hassanien), and The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609-0800 (Sundberg). Received for publication June 1, 1990. 36
Downloaded from vdi.sagepub.com at NEW YORK UNIV LIBRARY on April 24, 2015
Genital bovine papillomatosis
37
Figure 1. Section of a skin tumor from a cow. Bovine papillomavirus type 1 antigens are demonstrated by mouse monoclonal antibody AU-2 as brown-staining nuclei (arrowhead) within cells of the stratum granulosum of the epidermis. Immunoperoxidase stain, light green counterstain, 400 x .
thick) and immediately fixed in cacodylate-buffered 2% glutaraldehyde for 3 hr and postfixed in 1% osmium tetroxide.
Tissues were processed and sectioned routinely, stained with uranyl acetate and Reynold’s lead citrate, and examined in an electron microscope operated at 60 kV. Immunohistochemistry. Serial sections of the tumors were deparaffinized through a series of xylenes and graded ethanols. Nonspecific protein binding was blocked by incubation in 10% ovalbumin.9 A rabbit polyclonal antibody directed against papillomavirus group-specific antigensa was used as the primary antiserum. Affinity-purified rabbit IgGa was used as a negative control serum. A series of seven mouse MAbs 4 with various specificities (AU-l, -2, -3, -4, -5, -6, and 1H8) and normal mouse serum (negative control) were also used. The avidin-biotin complex (ABC) techniqueb was used to detect the bound antiserum, and diaminobenzidinec was used as the chromogen. Sections were counterstained with light green. Sections of bovine cutaneous fibropapillomas, confirmed by restriction analysis to contain either BPV-1 or BPV-2, were used as positive controls.
Results Histopathology. Microscopic features of all 3 tumors were identical. There was marked proliferation of the epithelium as well as proliferation of dermal fibroblasts, giving the tumors a broad papillary pattern. All layers of the epidermis were markedly thickened. Prominent changes were observed within the stratum granulosum, where individual and clusters of cells had abundant clear cytoplasm, often containing large pleo-
morphic keratohyalinlike granules. These cellular changes are typical of what have been called koilocytes, clear cells, or pale cells, changes considered to be typical for productive papillomavirus infections.7 The dermis consisted of densely packed fibroblasts and dense irregular collagenous connective tissue. The morphologic patterns were typical of fibropapillomas of papillomavirus etiology. Immunohistochemistry. Cells within the stratum granulosum of the proliferating epidermis that were undergoing degenerative changes resembling koilocytosis contained dark brown-staining nuclei (Fig. 1) typical of positive reactions for papillomavirus antigens. The nuclear staining continued into the stratum corneum; however, the nuclear size increased, then became poorly defined as would be expected in this layer. Positive nuclear staining was detected in all serial sections stained with both the monoclonal and polyclonal antibodies. The AU-2 antibody, which distinguishes BPV-1 from BPV-2 at high dilutions, stained virioncontaining cells at dilutions of 1:2,000. The BPV-2 control sections were stained at 1:100 dilutions but not at higher dilutions. This verified that the papillomavirus in these tissues was BPV-1. Chorioallantoic membrane inoculation. Conspicuous turbidity and thickening were observed on the CAM S of the chicken embryos 4-5 days after inoculation with the tumor suspensions on all CAM passages. No discrete lesions were detected on the CAM S
Downloaded from vdi.sagepub.com at NEW YORK UNIV LIBRARY on April 24, 2015
38
Elzein et al.
at any passage. Thin-section electron microscopy of infected CAMS did not reveal the presence of viruslike particles. Thin-section electron microscopy. Viruslike particles, 30-35 nm in diameter, were observed within nuclei of cells in the stratum granulosum in multiple sections taken from tumors of all 3 cattle. The particles formed large crystalline arrays.
been utilized effectively for prevention but not for treatment. Artificial insemination has virtually eliminated this disease in North America in herds using this breeding system. Acknowledgements This work was supported in part by grant number 2174 from the Council for Tobacco Research (JPS). We thank Dr. A. B. Jenson for graciously providing the monoclonal antibodies used in this study, Ann Higgins for her technical expertise in immunohistochemistry, and Mr. P. Prentis for the electron microscopy.
Discussion This paper describes the first case of genital bovine fibropapillomatosis in the Kingdom of Saudi Arabia and the Gulf Region and provides evidence that the Sources and manufacturers lesions were caused by a papillomavirus. Methods for the detection and prevention of this disease are needed a. DAKP Corp., Santa Barbara, CA. b. Vector Chemicals, Burlingame, CA. to prevent widespread dissemination as Saudi Arabia c. Sigma Chemical Corp., St. Louis, MO. begins to expand its cattle production. References The etiologic agent of genital fibropapillomas in cattle is generally considered to be BPV-1 or -2. 7 Molec- 1. Bruner DW, Gillespie JH: 1973, Hagan’s infectious diseases of ular studies are usually required to make a definitive domestic animals, 6th ed., pp. 1311-1314. Cornell University diagnosis. Immunohistochemistry and electron miPress, Ithaca, NY. croscopy are 2 generally available techniques for con- 2. Coggin JR, zur Hausen H: 1979, Workshop on papillomaviruses and cancer. Cancer Res 39:545-546. firming a papillomavirus etiology but are not adequate 3. Lancaster WD, Olson C: 1978, Demonstration of two distinct for typing. Panels of MAbs directed against various classes of bovine papilloma virus. Virology 89:371-379. BPV-1 epitopes can now be used to type or semitype 4. Lim P, Jenson AB, Cowsert L, et al.: 1990, Distribution and specific identification of papillomavirus major capsid protein epivarious papillomaviruses in tissue sections4 and were topes by immunocytochemistry and epitope scanning of synthetic useful in the cases described here for confirming a peptides. J Infect Dis 162: 1263-1269. BPV-1 etiology. The most accepted method of papil5. Pfister H: 1987, Papillomaviruses. General description, taxonlomavirus typing remains viral (low molecular weight) omy, and classification. In: The Papovaviridae, ed. Salzman NP, DNA extraction, restriction endonuclease digestion, Howley PM, vol. 2, The papillomaviruses, pp. l-38. Plenum Press, New York, NY. and high-stringency Southern blot analysis with appropriate controls. However, frozen tissues are re- 6. Sundberg JP: 1987, Animal models for papillomavirus research. Contrib Oncol 24: 11-38. quired for this approach. Immunohistochemical tech7. Sundberg JP: 1987, Papillomavirus infections in animals. In: niques provide an alternative when frozen tissues are Papillomaviruses and human diseases, ed. Syrjanen K, Gissmann not available or when shipping conditions preclude L, Koss LG, pp. 40-103. Springer-Verlag, Heidelberg. proper handling of such specimens. Information on 8. Sundberg JP: 1990, Bovine papillomaviruses. In: Diagnostic veterinary virology: a practitioner’s guide, ed. Castro AE, Heuschele papillomavirus type can then be utilized to develop WP (in press). Williams and Wilkins, Baltimore, MD. methods of prevention or treatment. 9. Sundberg JP, Junge RE, Lancaster WD: 1984, ImmunoperoxiMany approaches are available for the prevention dase localization of papillomaviruses in hyperplastic and neoof bovine genital fibropapillomatosis. The best method plastic epithelial lesions of animals. Am J Vet Res 45: 1441-1446. is to maintain a clean herd.8 Autogenous vaccines have
Downloaded from vdi.sagepub.com at NEW YORK UNIV LIBRARY on April 24, 2015
