Main topic : Animal Health

DETECTION OF BOVINE PAPILLOMAVIRUS TYPE 1, 2 AND 13 DNA IN EQUINE SARCOIDS USING TWO DIFFERENTS METHODS

DE PAOLIS L. ¹, ZOCCOLA R. ², DE CIUCIS C. ¹, MARTINI I. ¹, PORCELLATO I. ³, COCUMELLI C. ⁴, MECOCCI S. ³, PIGOLI C. ⁵, MECHELLI L. ³, GIALLETTI R. ³, GIBELLI L. ⁵, PASSERI B. ⁶, GORIA M. ², CAPPELLI K. ³, ELENI C. ⁴, GHELARDI A. ⁷, RAZZUOLI E. ¹

¹ National Reference Center of Veterinary and Comparative Oncology (CEROVEC), Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, Genova, Italy; ² Department of Biotechnology, Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, Torino, Italy; ³ Department of Veterinary Medicine, University of Perugia, Perugia, Italy; ⁴ Istituto Zooprofilattico Sperimentale del Lazio e della Toscana, Roma, Italy; ⁵ Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna, Brescia, Italy; ⁶ Department of Veterinary Medicine, University of Parma, Parma, Italy; ⁷ Azienda Usl Toscana Nord-Ovest, UOC Ostetricia e Ginecologia, Ospedale Apuane, Massa, Italy

Introduction
Equine sarcoid is the most commonly diagnosed skin neoplasm in horses, representing up to 90% of all skin tumors. Clinically, it occurs as a fibroblastic, wart-like and locally aggressive lesion, often present as single or multiple tumors (1). Although sarcoids are not fatal, their high incidence, resistance to therapy and tendency to recurrence, may impact the welfare and value of the horse, eventually leading to the withdrawal of the animal from use. This tumor’s etiology is multifactorial; however, the association between genetic factors and bovine papillomaviruses (PVs) types 1, 2 and 13 (BPV1, BPV2 and BPV13) seems to underlie the development of the tumor (2,3). To date, PCR-based methods are the most commonly used for detecting BPV viral DNA. Among these methods, droplet digital Polymerase Chain Reaction (ddPCR) has been recently proposed as a direct and sensitive technique for quantifying nucleic acids, since it yields absolute concentrations without the requirement of a standard calibration curve, thus rendering the detection process faster and more sensitive (4). The aim of the study was to assess BPV 1, 2 and 13 DNA in equine sarcoids through ddPCR and to compare the results obtained with those yielded by real time qPCR.

Methods
In order to investigate the presence of BPV types 1, 2 and 13, two different PCR-based methods-real time qPCR and ddPCR, respectively-were applied. Sixtyfour Formalin-Fixed Paraffin-Embedded (FFPE) samples of equine sarcoids were retrieved from the archives of the Universities of Parma and Perugia and the Experimental Zooprophylactic Institutes of Lazio-Toscana and Lombardia-Emilia Romagna. DNA extracted using QIAamp DNA Mini Kit (Qiagen) was submitted to real-time PCR targeting the L1 region, using primers and probes specifically developed. The same set of primers and probes was used to test samples by Droplet-PCR. Serial dilutions of extract DNA were set up for ddPCR analyses. Five microliters of each dilution were added to the PCR mixes prepared with the ddPCR Supermix for Probes (No dUTP) (Biorad), analyzed with the QX200 Droplet Generator&Reader system (Biorad) and produced data were processed with QX Manager Software Standard Edition, Vs 1.2 (Biorad).

Results
Real time qPCR and ddPCR showed identical results. In detail, BPV1 DNA was found in 89.1% of samples and BPV2 on 21.9%. In particular, 47 lesions were positive for BPV1 and 4 for BPV2 DNA. Moreover, 10 out of 64 samples showed copresence of both BPV1 and BPV2 DNA. All samples tested by both methods, resulted negative for BPV13.

Conclusion
ddPCR appeared to be an efficacious means for detecting BPV viral DNA, with results comparable to those obtained through real time qPCR. Preliminary results obtained suggested that sarcoids from Italian horses are most often caused by BPV 1. Moreover, despite its moderate size, the sample could be considered representative enough to suggest a low occurrence of BPV13 in Italy. However, future tests will be needed in order to estimate of the prevalence and distribution of BPV13 infections in European horse populations.