Detection of bovine papillomavirus DNA in sarcoid-affected and healthy free-roaming zebra (Equus zebra) populations in South Africa

Abstract

The endangered Cape mountain zebra (Equus zebra zebra) is protected in small numbers in a few isolated populations in South African game parks. Since 1995, sarcoid lesions appeared in zebras in two of the parks. This study was undertaken to investigate if bovine papillomavirus (BPV) is associated with sarcoids in these zebras. A conventional PCR, targeting the E5 ORF of BPV, and subsequent RFLP analysis were initially used to demonstrate the presence of BPV-1 and -2 DNAs in zebra sarcoid tumours. A rapid, sensitive and reliable real-time PCR to detect and distinguish between BPV-1 and -2 infections in zebras was developed. With this assay it was demonstrated that BPV-1 and -2 DNA (either single or mixed infections) are present in sarcoid tumour, healthy skin and blood of sarcoid-affected and healthy zebras from sarcoid-affected parks as well as in the blood of zebras from parks where no sarcoid has been observed before.

Introduction

The Cape mountain zebra (Equus zebra zebra) is one of the rarest mammals in the world (Penzhorn, 2003) and classified as endangered on the International Union for the Conservation of Nature and Natural Resources (IUCN) Red List 2006 (http://www.iucnredlist.org). They are protected in small numbers in a few isolated populations in South African game parks in their natural habitat. Of the three extant species of zebra in the family Equidae, genus Equus, subgenus Equus zebra, consists of two subspecies: the Cape mountain zebra, E. zebra zebra, from the Eastern and Western Cape Province, South Africa and Hartmann's mountain zebra, E. zebra hartmannae of Namibia and Angola. Cape mountain zebra populations have historically declined due to hunting, habitat loss, droughts and the interbreeding of the two subspecies. Although their numbers have slowly increased in recent years, the genetic diversity and the continued existence of Cape mountain zebras are of great concern as the uneven distribution of a few relatively large populations makes them still vulnerable (Novellie et al., 2002).

Not many infectious diseases are associated with zebras. They are resistant to African horsesickness (Barnard, 1993, Barnard, 1994, Lord et al., 1997), a viral disease of horses, and equine herpesvirus infections appear to be widespread in free-ranging zebras (Blunden et al., 1998, Borchers and Frölich, 1997, Borchers et al., 2006). The presence of antibodies against equine encephalosis virus (EEV) has also been shown (Barnard and Paweska, 1993). Although no evidence of infection with equine arteritis virus could be found in South Africa (Barnard and Paweska, 1993), antibodies were demonstrated in Burchell's zebras (Equus burchelli) from the Serengeti ecosystem (Borchers et al., 2005). Bovine papillomaviruses (BPV) are associated with sarcoids, the most common dermatological skin lesion in equidae (Goodrich et al., 1998, Jackson, 1936). Bovine papillomavirus types 1 and 2 (BPV-1 and -2) have been detected in sarcoid tumours of horses, donkeys and mules (Angelos et al., 1991, Bloch et al., 1994, Chambers et al., 2003b, Lancaster et al., 1977, Nasir et al., 1997; Nasir and Reid, 1999; Reid et al., 1994) and 2 cases were reported from captive zebras (Equus burchelli boehmi), 1 from Mexico and 1 from a private wild animal farm in WA State, United States of America (Löhr et al., 2005). In 1995, sarcoid lesions appeared in Cape mountain zebras in the Gariep Dam Nature Reserve, Free State Province (Nel et al., 2006) and in 1998 in the Bontebok National Park, Western Cape Province (Lange, 2004). An isolated case of a zebra that was euthanized due to the severity of sarcoid lesions was reported in 2004 in the Mountain Zebra National Park, Eastern Cape Province. Subsequently 4 more cases were reported (personal communication: Dr. Dave Zimmerman). Sarcoid lesions are the most common dermatological skin lesions in domestic equidae (Goodrich et al., 1998, Jackson, 1936). The term “equine sarcoid” was first used by Jackson in South Africa in 1936 to describe a distinctive fibroblastic neoplasm occurring in the skin of horses, donkeys and mules (Jackson, 1936). It was also used to distinguish the neoplasm from papilloma, fibroma and fibrosarcoma. Equine sarcoid is a locally aggressive, non-regressing, fibroblastic skin tumour, which does not produce infectious virions (Amtmann et al., 1980, Lancaster, 1981).

All papillomaviruses are classified in the family Papillomaviridae (Bernard, 2006). This large family of animal and human viruses generally infects epithelial cells causing hyperproliferative lesions known as warts, papillomas or condylomas. Typically these lesions are benign, self-limiting and spontaneously regress, although some are linked to malignancy such as the human papillomavirus 16 and 18 (WHO/IARC, 1995). Some types of papillomaviruses can also infect fibroblasts and induce fibro-epithelial tumours, including bovine papillomavirus-1 and bovine papillomavirus-2, which cause benign fibropapillomas in cattle (Nasir et al., 2007). All papillomaviruses except one are strictly species-specific, only known exception being naturally occurring cross-species infection of horses, donkeys and mules with BPV-1 and -2 (Nasir et al., 2007) in which they are associated with sarcoids (Campo, 2002, Lancaster and Olson, 1980, Nasir and Reid, 1999).

Both BPV-1 and -2 have a genome of 7900 bp of double stranded DNA and are composed of early (E) and late (L) genes which can be divided into several open reading frames: viral replication (E1), regulation of transcription (E2), coding for cytoplasmic proteins (E4), transforming proteins (E5, E6 and E7) as the early genes and L1 and L2 coding for capsid proteins as late genes (Chambers et al., 2003a, Nasir and Campo, 2008). Studies concerning gene expression have largely focused on expression of the major BPV early genes, E2, E5, E6 and E7 (Bogaert et al., 2007, Yuan et al., 2007).

Equine sarcoids appear in different clinical entities and can be classified into six distinct clinical types (Knottenbelt, 2005). Histopathological examination is often required to confirm diagnosis and to distinguish them from other skin lesions (Goodrich et al., 1998, Jackson, 1936, Martens et al., 2000). PCR techniques are suitable for the investigation of papillomavirus-associated benign and malignant lesions (Bloch et al., 1994) and PCR has been used to elucidate the role of bovine papillomavirus in the induction of equine sarcoids. Most equine sarcoids are found to contain viral sequences of either BPV-1 or -2 (Amtmann et al., 1980, Angelos et al., 1991, Bogaert et al., 2005, Carr et al., 2001a, Carr et al., 2001b, Lancaster and Olson, 1980, Lancaster, 1981, Otten et al., 1993, Ragland et al., 1970, Reid and Smith, 1992, Reid et al., 1994, Teifke and Weiss, 1991, Trenfield et al., 1985, Yuan et al., 2007). Several workers have used primers specific for the amplification of a 244 bp and 247 bp region of the E5 open reading frame (ORF) of BPV-1 and -2, respectively (Bloch et al., 1994, Carr et al., 2001b, Martens et al., 2001, Otten et al., 1993, Teifke et al., 1994). Restriction fragment length polymorphism (RFLP) is then generally used to differentiate between BPV-1 and -2 as the BPV-1 amplified product contains a BstX1 restriction site which is absent in BPV-2 (Bloch et al., 1994, Teifke and Weiss, 1991). The PCR assay has been used successfully to demonstrate BPV DNA in up to 100% of examined equine sarcoids in several studies (Carr et al., 2001b, Martens et al., 2001, Otten et al., 1993, Teifke et al., 1994). Recently, quantitative real-time PCR assays have been developed and used to determine viral load and the expression of BPV E2, E5, E6 and E7 genes in equine sarcoids and inflammatory skin conditions (Bogaert et al., 2007, Yuan et al., 2007).

The purpose of this study was to determine if BPV is also present in sarcoid in free-roaming zebras by using PCR and RFLP analysis, and to develop a real-time PCR diagnostic method to detect and distinguish between BPV-1 and -2.