Papillomavirus infection of roe deer in the Czech Republic and fibropapilloma- associated levels of metallothionein, zinc, and oxidative stress

The present study aimed at the aetiological diagnosis of skin tumours of roe deer (Capreolus capreolus) in the Czech Republic. A total of 33 roe deer specimens showing skin masses were sampled for histopathology, virus detection and identification using polymerase chain reaction (PCR) and deoxyribonucleic acid sequence (DNA) analysis, and to investigate fibropapillomaassociated levels of metallothionein, zinc and oxidative stress in 2012 and 2013. Ticks (Ixodes ricinus) and deer keds (Lipoptena cervi) were also collected and pooled from sampled game specimens. Ticks found on dogs in hunting grounds under study were pooled into another sample. Skin tumours, ranging from 2 to 10 per inspected specimen and 2 to 5 cm in size, were classified as fibropapillomas by histopathology. All 33 viral-infection-suspected skin samples from roe deer in South Moravia and South Bohemia were PCR positive. The nucleotide sequences of PCR products were 100% homologous to the Western roe deer papillomavirus 1 isolate CcPV-1. Ticks and deer keds from positive roe deer and ticks from dogs were PCR and DNA sequence positive for the roe deer papillomavirus. Viral DNA was also demonstrated in one blood sample from a roe deer female. Differences in metallothionein, zinc, taurine and electrochemical index among samples from lesions and normal skin of affected roe deer and negative controls from papillomavirus-non-infected animals were non-significant. While we have demonstrated circulation of specific roe deer papillomavirus in the Czech Republic that results in multiple fibropapillomatous skin tumours, many ecological and epidemiological issues of this wildlife disease still remain unanswered. Capreolus capreolus, Lipoptena cervi, Ixodes ricinus, papillomatosis, skin tumours, hunting dog The roe deer (Capreolus capreolus) is an important native game ungulate species found throughout most habitats of Europe (Pikula et al. 1997), with estimated spring population and annual bag records in the Czech Republic amounting to 310 000 and 120 000 specimens, respectively. Importantly, annual mortality reports stand at nearly 40 000 in this central European country. In an analysis of mortality of 1 606 roe deer specimens in the Czech Republic, it was found that dietary, parasitic, infectious, toxic, and traumatic causes were responsible for 46, 41, 5, 4, and 4% of deaths, respectively (Sterba et al. 1997). While infections play a minor role in roe deer mortality, a novel roe deer papillomavirus infection endemic to the Carpathian basin area of Hungary, Austria and Croatia has been recognised (Erdélyi et al. 2009). Phylogenetic analysis placed the roe deer papillomavirus ACTA VET. BRNO 2015, 84: 105–111; doi:10.2754/avb201584020105 Address for correspondence: Prof. MVDr. Jiri Pikula, Ph.D. Department of Ecology and Diseases of Game, Fish and Bees Faculty of Veterinary Hygiene and Ecology University of Veterinary and Pharmaceutical Sciences Brno Palackého tř. 1/3, 612 42 Brno, Czech Republic Phone: +420 541 562 655 Fax: +420 541 562 657 E-mail: [email protected] http://actavet.vfu.cz/ within the Delta-papillomavirus genus (Erdélyi et al. 2008). Fibropapillomatous skin tumours are easy to recognise (Erdélyi et al. 2009), and, to date, the disease has also been reported from Slovak and Czech regions bordering with Hungary and Austria. Where prevalent, it may endanger the economics of trophy hunting because of the repulsive appearance of affected animals. However, there is still a lack of proper diagnosis based on virus identification and genetic analysis from these two new countries of disease occurrence. The latest research indicates that metallothioneins (MTs) play a role in many pathophysiological processes including viral infections connected with a tumour disease (Theocharis et al. 2003; Krizkova et al. 2009). Interestingly, apart from metal ion homeostasis, detoxification, protection against oxidative damage, and cell proliferation and apoptosis, metallothionein isoforms have been shown to be involved in several aspects of the carcinogenic process, cancer development and progression (Cherian et al. 2003; Theocharis et al. 2003; Krizkova et al. 2008). Elevated MT concentrations in cancer cells are probably related to their increased proliferation and protection against apoptosis (Krizkova et al. 2009). Metallothioneins, a group of low-molecular weight, cysteine-rich intracellular proteins, control the cellular zinc ion levels and proper intracellular Zn(II) concentration maintains the fragmentation of DNA associated with caspase-3 activity. As the expression of MTs has been linked with tumour spread, it may be a potential prognostic marker for certain tumours (Dutsch-Wicherek et al. 2008). Moreover, the metal ion detoxification function of MTs may interfere with chemotherapy (Theocharis et al. 2003; Eckschlager et al. 2009). Extensive observations suggest that oxidative stress, chronic inflammation, and cancer are closely linked (Reuter et al. 2010). Taurine, a major cytosolic free amino acid of leukocytes, scavenges for reactive oxidants such as hypohalous acids HOCl and HOBr generated by activated neutrophils at sites of inflammation (Nejdl et al. 2013). Electrochemical voltammetry is a non-specific method of evaluation of oxidative stress in biomedicine that allows for comparison of overall antioxidant activity associated with the pathology under study (Pohanka et al. 2011, 2012). As viral aetiology of fibropapillomatous skin tumours of roe deer from the Czech Republic has not yet been confirmed and nothing is known about metallothionein and zinc concentrations and oxidative stress in this type of tumour in wildlife, the objective of the present study was to detect and identify the roe deer papillomavirus in suspect samples collected in Czech hunting grounds and investigate fibropapilloma-associated concentrations of metallothionein, zinc, taurine, and the electrochemical index. Furthermore, the role of ectoparasites as potential vectors of the roe deer papillomavirus was examined. Materials and Methods Laboratory diagnostics of skin tumours included histopathology, PCR and DNA sequence analysis. Paired samples of normal skin and fibropapillomatous tumours were collected from a total of 33 roe deer specimens (15 males and 18 females) from South Bohemia and South Moravia (Czech Republic) in 2012 and 2013. These roe deer (Capreolus capreolus) specimens were killed during the regular hunting season and aged from 3 to 8 years. Other 10 specimens without clinical skin disease, younger than 1 year and with a sex ratio of 1:1 were sampled to obtain the negative control of skin. Blood was also sampled by cardiac puncture within half an hour of killing the specimens used in the study. Each skin and fibropapilloma sample was divided into parts for histopathology, virus detection and identification, and measurements of metallothionein and zinc concentrations, oxidative stress and amino acids. Fresh samples for histopathological examination were placed in 10% buffered formalin, treated using a routine histological technique and embedded in paraffin. Sections of 5 μm thickness were made from the paraffin blocks, and these were stained with haematoxylin and eosin. The other parts were stored at -20 °C until analysis. The DNA was extracted from approximately 20 mm3 using a commercial kit (DNeasyR Blood & Tissue Kit, Qiagen GmbH, Germany) for polymerase chain reaction and sequence analysis. Ticks (Ixodes ricinus) and deer keds (Lipoptena cervi) were also collected and pooled from individual sampled game specimens. A total of 83 ticks found on dogs used in hunting grounds under study were pooled into another 106