Intralesional bovine papillomavirus DNA loads reflect severity of equine sarcoid disease R. HARALAMBUS, J. BURGSTALLER † , J. KLUKOWSKA-RÖTZLER, R. STEINBORN ‡ , S. BUCHINGER § , V. GERBER and S. BRANDT* ¶ Equine Clinic, VetSuisse Faculty, University of Berne, Laenggass-Strasse 120, CH-3012 Berne, Switzerland; † Department of Biotechnology in Animal Production, IFA-Tulln, Konrad-Lorenz-Strasse 20, A-3430 Tulln, Austria; ‡ Vetomics Core Facility, University of Veterinary Medicine, Veterinaerplatz 1, A-1210; § Faculty of Computer Science, University of Vienna, Lenaugasse 2/8, A-1080; and ¶ Equine Biotechnology Unit, University of Veterinary Medicine, Veterinaerplatz 1, A-1210 Vienna, Austria. Keywords: horse; bovine papillomavirus; equine sarcoid; quantitative PCR; viral DNA load Summary Reasons for performing study: Sarcoids are nonmetastasising, yet locally aggressive skin tumours that constitute the most frequent neoplasm in equids. Infection by bovine papillomaviruses types 1 and 2 (BPV-1, BPV-2) has been recognised as major causative factor in sarcoid pathogenesis, but a possible correlation of intralesional virus load with disease severity has not been established thus far. Hypothesis: Given the pathogenic role of BPV-1 and BPV-2 in sarcoid disease, we suggest that intralesional viral DNA concentration may reflect the degree of affection. Methods: Severity of disease was addressed by recording the tumour growth kinetics, lesion number and tumour type for 37 sarcoid-bearing horses and one donkey. Viral load was estimated via quantitative real-time PCR (qPCR) of the E2, E5, L1 and L2 genes from the BPV-1/-2 genome for one randomly selected lesion per horse and correlated with disease severity. Results: Quantitative PCR against E2 identified viral DNA concentrations ranging from 0–556 copies/tumour cell. Of 16 horses affected by quiescent, slowly growing single tumours or multiple mild-type lesions, 15 showed a viral load up to 1.4 copies per cell. In stark contrast, all equids (22/22) bearing rapidly growing and/or multiple aggressive sarcoids had a viral load between 3 and 569 copies per cell. Consistent results were obtained with qPCR against E5, L1 and L2. Conclusions: While tumours of the same clinical type carried variable virus load, confirming that viral titre does not determine clinical appearance, we identified a highly significant correlation between intralesional viral load and disease severity. Potential relevance: The rapid determination of BPV viral load will give a reliable marker for disease severity and may also be considered when establishing a therapeutic strategy. Introduction Bovine papillomaviruses (BPV) are well studied animal viruses that consist of a nonenveloped protein capsule harbouring a circular, double-stranded DNA genome coding for early (E) functional and late (L) structural proteins. In cattle, BPV types 1 and 2 (BPV-1, BPV-2) mostly induce transient epithelial papillomas that rarely undergo malignant transformation (Campo et al. 1992). In addition, BPV-1 and to a lesser extend BPV-2 chiefly contribute to the development and maintenance of nonmetastasising, yet locally aggressive skin tumours in equids (Nasir and Reid 2006). These lesions, termed sarcoids, are mostly persistent and therapy-resistant, and tend to recur following treatment. With an incidence ranging from 12.5 to 67% of all equine cancers, they represent the most common neoplasm in horses (Sullins et al. 1986). Sarcoids can develop at any body site as a single tumour or as multiple lesions of various types (Scott and Miller 2003). According to their gross appearance, sarcoids are classified as occult, verrucous, nodular, fibroblastic, mixed or malevolent lesions. Occult sarcoids represent the mildest form of disease and consist of slowly growing, hairless circular to oval skin areas. They may progress to a wart-like, verrucous tumour type of rough, hyperkeratotic appearance. Nodular sarcoids represent a more advanced grade of disease and are typically recognised as firm subcutaneous nodules lying under apparently intact skin, whereas aggressive fibroblastic lesions have a fleshy appearance with pronounced ulceration and serum exudation. Mixed sarcoids contain any of the described types and often appear as confluent aggregates of fibroblastic, nodular and verrucous lesions. Malevolent sarcoids constitute the most severe type of disease as they consist of multiple, partly ulcerative nodules that equally extend to local lymph nodes. Trauma is regarded as a cofactor in sarcoid development as it contributes to tumour onset and also the progression from mild to more pronounced affection (Knottenbelt et al. 1995; Carr et al. 2001a; Knottenbelt 2005). Clinically, disease severity can be evaluated on the basis of the sarcoid type(s), the number of lesions and tumour growth characteristics (slow or rapid, primary or recurrent, skin-restricted or invasive). Early virus inoculation experiments (Olson and Cook 1951; Ragland and Spencer 1969), Southern blot (Amtmann et al. 1980; Lancaster 1981) and PCR revealing viral DNA in the vast majority of investigated lesions (Otten et al. 1993; Bloch et al. 1994; Carr et al. 2001a,b; Martens et al. 2001a,b; Bogaert et al. 2005, 2008) and also intact skin (Carr et al. 2001b; Bogaert et al. 2005, 2008) *Author to whom correspondence should be addressed. [Paper received for publication 05.05.09; Accepted: 31.07.09] EQUINE VETERINARY JOURNAL 327 Equine vet. J. (2010) 42 (4) 327-331 doi: 10.1111/j.2042-3306.2010.00078.x © 2010 EVJ Ltd