Virus Research 144 (2009) 315–317 Contents lists available at ScienceDirect Virus Research journal homepage: www.elsevier.com/locate/virusres Short communication The detection of Bovine Papillomavirus type 1 DNA in flies Margaret Finlay a , ZhengQiang Yuan a , Faith Burden b , Andrew Trawford b , Iain M. Morgan a , M. Saveria Campo a , Lubna Nasir a,∗ a Institute of Comparative Medicine, Faculty of Veterinary Medicine, University of Glasgow, Bearsden Road, Glasgow G61 1QH, Scotland, UK b The Donkey Sanctuary, Sidmouth, Devon EX10 0NU, UK article info Article history: Received 1 April 2009 Received in revised form 22 April 2009 Accepted 22 April 2009 Available online 3 May 2009 Keywords: Bovine Papillomavirus Disease transmission Equine sarcoid Flies abstract BPVs are double stranded DNA viruses that can infect several species other than the natural host, cattle, including equids. In equids, BPV-1, and, less commonly BPV-2, infection gives rise to fibroblastic tumours of the skin. Whilst a causal relationship between BPV-1/2 and equine sarcoids is now well established, how the disease is transmitted is not known. In this study we show BPV-1 DNA can be detected in flies trapped in the proximity of sarcoid-affected animals. Sequence analysis of the BPV-1 LCR from flies indicates that flies harbour BPV-1 LCR sequence variants II and IV which are commonly detected in equine sarcoids. These data suggest that flies may be able to transmit BPV-1 between equids. © 2009 Elsevier B.V. All rights reserved. Sarcoids affect all equids including horses, donkeys, mules and zebras. They are locally invasive fibroblastic skin tumours and represent the most common equine neoplasm with reported preva- lence rates ranging from 12.9 to 67% of all equine tumours (Sullins et al., 1986). It is now well established that Bovine Papillomavirus (BPV) (type 1 and, less commonly type 2) is involved in the patho- genesis of sarcoids (Amtmann et al., 1980; Nasir and Reid, 1999; Carr et al., 2001a; Chambers et al., 2003; Nasir et al., 2007; Yuan et al., 2007). The vast majority of sarcoid tumours harbour episo- mal BPV genomes localised to the fibroblast nuclei (Lancaster, 1981; Teifke et al., 1994; Löhr et al., 2005). Viral genes and proteins can be detected in sarcoids indicating an active infection (Carr et al., 2001b; Chambers et al., 2003; Borzacchiello et al., 2008). Whilst viral parti- cles have not been detected, Brandt et al. (2008) have demonstrated BPV-1 virus-like structures in a subset of sarcoids using immuno- capture PCR. This is the first evidence for the presence of putative intralesional BPV virions in the disease. Currently, little is known about how the disease is transmitted between animals. Early studies showed that transient sarcoid-like lesions can be induced via inoculation with sarcoid extract and purified virus (Olson and Cook, 1951; Ragland and Spencer, 1969; Lancaster et al., 1977), however no further studies have addressed this important question. We have recently shown that equine sar- coids are most often associated with specific BPV-1 variants which are not present in BPV-1 infections in cattle (Nasir et al., 2007), ∗ Corresponding author. Tel.: +44 141 330 5731; fax: +44 141 330 5602. E-mail address: l.nasir@vet.gla.ac.uk (L. Nasir). suggesting that there is horse-to-horse transmission. We have also published data supporting donkey-to-donkey transmission of BPV- 1 by sequence analysis of BPV-1 long control region (LCR) (Nasir and Campo, 2008). Sarcoid lesions often occur at wound sites or sites predisposed to trauma and this has led to the hypothesis that flies may act as vectors in disease transmission between animals. The aim of this study was to assess whether BPV-1 DNA can be detected in flies trapped in the proximity of sarcoid-affected animals. Adhesive, chemical-free paper-strip traps (Rentokil Fly Killer Strips, UK) were used to catch flies at several locations at the Don- key Sanctuary, Devon, at three different time points over the course of the 2008 summer season (June, August and September) (Table 1). The traps were left in position for up to 2 weeks and then individ- ually bagged and stored at 4 ◦ C until required. Flies were removed from traps and batched together into species groups. In June 2008, Isohelix DNA buccal swabs (Cell Projects, UK) were used to collect surface cells from sarcoid lesions present on the animals housed at the fly trap locations. The swabs were then sealed in sterile bags and stored at 4 ◦ C. DNA was extracted from flies and sarcoid swabs using the Qiagen DNeasy Blood and Tissue Kit (Qiagen, UK). To confirm successful fly DNA isolation, samples were subjected to PCR ampli- fication using fly mitochondrial 16s rDNA gene primers; forward primer FlyF: 5 ′ TTACGCTGTTATCCTAA3 ′ and reverse primer FlyR: 5 ′ CACCTGAACAAAAACAT3 ′ (Simon et al., 1994). The reactions were performed using 100 ng of the extracted DNA and 24 l of PCR-mix consisting of 1 l (20 pmol) from each primer; 200 M of each dNTP (Invitrogen Life Sciences, UK); 1.25 units of Platinum Taq DNA poly- merase (Invitrogen); 1× PCR buffer (20 mM Tris–HCl pH 8.4 and 0168-1702/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2009.04.015