BPV is a viral agent forming benign lesions within cutaneous or mucosa epitheliums in different areas of the body (Maclachan et al. 2017). Cutaneous lesions have different morphologies such as filiform, peduncle, atypical or squamous (Lunardi et al. 2013; Daudt et al. 2018). On the other hand, the virus causes carcinomas in vesicaurinaria, genital and gastrointestinal channel mucosas (Borzacchiello and Roperto 2008; Lunardi et al. 2013; Roperto et al. 2013).
The agent causes full-scale economical losses leading to lesions of various size and structure in animals such as goats, cattle and sheep grown for milk and meat production (Tan et al. 2012; Araldi et al. 2017; Daudt et al. 2018). Papillomas appearing on teats of animals especially in milker herds makes milking impossible and decreases production (Campo 2002). In addition, injuries appearing on teat skin dependent on papillomatosis might make the area predisposed against secondary infections or might cause mastitis (Campo 2002; Zhu et al. 2019).
Detection of papillomaviruses could be done molecularly by using degenerate primers (FAP59/FAP65 - MY09/MY11) designed for gene areas coding L1, L2, E6 and E7 proteins (Ogawa et al. 2004). In samples of cutaneous papillomatosis, during the studies carried out for molecular detection of BPV using FAP59/FAP64 degenerate primers, the rate of prevalence was found as between 54-100% (Ogawa et al. 2004; Silva et al. 2013a; Dağalp et al. 2017). In this study that we carried out using FAP59/64 degenerate primers, viral genome detection was performed in all teat papilloma samples (100%). This result is such as to support the results obtained from other studies. No positivity was detected in the samples using the other degenerate primer set. During the studies, Silva et al. (2013b) stated that BPV type specific primers were more sensitive than degenerate primers and degenerate primers fail the recognize in papilloma samples with mixed types. Silva et al. (2013a) found that the specifity of FAP59/64 and MY09/MY11 degenerate primers was lower than type specific primers, it couldn’t detect some viral types (for example BPV-4, 9) and the reason for this was that it was designed for human papillomavirus identification (HPV). The difference in BPV infection prevalence is believed to have occurred for these reasons.
Sequencing analysis was performed on L1 gene PCR products amplified from the collected samples and revealed that there were four types of BPV (BPV-2, BPV-6, BPV-8 and BPV-10). Nucleotide similarities of BPV isolates detected in teat tissue in this study are shown in Table 2. The phylogenetic analysis conducted using the coding sequence of the L1 gene revealed that the tree was divided into five main groups; Xipapillomavirus 1, Dyoxypapillomavirus 1, Epsilonpapillomavirus 1, Deltapapillomavirus and new putative papillomavirus. Five of the nucleotide sequences isolated in this study, (Turkey_BUR1_Teat_BPV, Turkey_BUR2_Teat_BPV, Turkey_BUR4_Teat_BPV, Turkey_BUR5_Teat_BPV, Turkey_BUR7_Teat_BPV) clustered into the Xipapillomavirus 1, Turkey_BUR6_Teat_BPV clustered into the Epsilonpapillomavirus 1, Turkey_BUR3_Teat_BPV clustered into the Deltapapillomavirus (Figure 1). According to the phylogenetic tree constructed, BPV isolates were not grouped according to the geographic distribution. At the end of phylogenetic analysis, the obtained isolates showed genetic closeness to those obtained in Brazil, Turkey, Croatia and China.
In previous studies on the histopathological examination of papilloma cases; acanthosis, hyperplasia of the spinal epithelial layer, koilocytosis, hypergranulosis, hyperkeratosis, parakeratosis, papillomatosis, transformed fibroblasts and vacuolar degenerations of the stratum spinosum of the dermis have been reported (Anjos et al. 2010; Marins and Ferreira 2011; Tan et al. 2012; Timurkan and Alcigir 2017). While hyperkeratosis and acanthosis are common findings in papilloma cases, in addition to epidermal proliferations increased connective tissue mainly fibroblasts are the main basic findings in fibropapillomas (Scagliarini et al. 2016). In histopathological studies on teat papillomas of BPV, Jelinek and Tachezy (2005) encountered fibropapillomas containing many koilocytes, neoplastic fibroblasts in the dermis, and acanthosis and orthokeratotic hyperkeratosis in the epidermis. Maeda et al. (2007) identified structures characterized by epithelial hyperplasia, acanthosis and hyperkeratosis, but they could not detect fibropapilloma. Hatama et al. (2009) found epidermis hyperplasias with hyperkeratosis and marked acanthosis. Hatama (2012) stated that they observed epithelial hyperproliferation with normal dermal appearance. Beytut (2017), on the other hand, observed widespread epidermal hyperplasia, acanthosis and hyperkeratosis, as well as an increase in connective tissue in the dermis. However, koilocytes have seen balloon-like degeneration, pycnotic and nuclear fragmentation containing dense chromatin. In this study, our histopathological examination findings, which we mentioned in detail above, in teat lesions were similar to the results of other researchers. At the histopathological examination, proliferations were observed in the epidermis and dermis, in fibropapilloma cases while only epidermal proliferations together with excessively thickened in the keratin layer notices in papilloma cases. Acanthosis and parakeratosis were evident in the keratin layer. Spongiosis and degenerations were frequently observed in the squamous cells of the epidermis. Koilocytes were frequently seen in the stratum spinosum and granulosum. Rete peg formations characterized by severe proliferations in to the dermis towards the epidermis were common findings. The connective tissue which mostly composed of fibroblast and collagen fiber bundles, extended in different directions. In some cases, marked proliferations were observed in the dermis, and such cases were evaluated as fibropapilloma.
In our study, positive immunoreactions were observed in the epidermis and keratin layer in the sections immunohistochemically stained with BPV antibody. In a few cases, a positive reaction was also detected in the capillaries of the dermis. In previous studies, intranuclear or cytoplasmic antigen-antibody reaction was revealed by immunohistochemical method in basophilic intranuclear inclusion bodies in the stratum granulosum layer of the epidermis, endothelial cells of vessels, mesenchymal, horny and granular cells and corneal layer in papilloma cases (Jelinek and Tachezy 2005; Maeda et al. 2007; Catroxo et al. 2013; Beytut 2017; Babu et al. 2020). In fibropapillomas, they reported that positive reactions in the cytoplasm of fibroblasts in the dermis, in different layers of the epidermis, and in the cell nuclei of the basal layer and nuclei around the blood vessels (Jelinek and Tachezy 2005; Maeda et al. 2007). In our study, findings and results determined by immunohistochemical diagnosis in teat papillomas were in parallel with the results of the other researchers.
In the previous studies, the detection rate of viral agents by electron microscopy was between 0-100% was reported. Many researchers (Brobst and Hinsman 1966; Pulley et al. 1974; Taichman and LaPorta 1987) stated that BPV first settles in the cell nuclei of the cells of stratum spinosum layer of the epidermis. They caused degeneration at the nuclei of the cells in the stratum granulosum and stratum corneum, and remaining viruses spread. In addition, PVs can only do vegetative replication in epithelial cells, progeny virus cannot be obtained in fibroblasts, and therefore ends with biological death. It was concluded that this difference in prevalence values was due to the fact that the researchers examined the regions other than the areas where the agent replicated, or they tried to detect the virus before the spread of the progeny viruses. In this study, the presence of virus was detected in all lesions as a result of electron microscopical examinations performed on the samples of teat papilloma lesions selected from the positive cases with BPV type specific primers.
As a result, in this study, serotypes of BPV causing teat papillomas in cattle were detected. In order to provide BPV identification molecularly, consensus primers were to be used before specific primers, which would then prevent loss of time and material. In diagnosis of the agent in teat lesions depending on BPV infection, PCR, histopathology, immunohistochemistry and electron microscopy applications were parallel to one another. It would be useful to take biosafety measures in cattle breeding, to search for factors causing the teat tissue to be predisposed for BPV infection, to detect the most common BPV types and to develop standard commercial vaccines for teat papillomas for these types.