Bovine papillomaviruses (BPVs) are oncogenic DNA viruses, which mainly induce benign lesions of cutaneous and/or mucosal epithelia in cattle. Thirteen (BPV 1–13) different viral genotypes have been characterized so far. BPVs are usually species-specific but BPV 1/2 may also infect equids as well as buffaloes and bison and cause tumors in these species. BPV-induced benign lesions usually regress, however occasionally they develop into cancer particularly in the presence of environmental carcinogenic co-factors. The major transforming protein of BPV is E5, a very short hydrophobic, transmembrane protein with many oncogenic activities. E5 contributes to cell transformation through the activation of the cellular β receptor for the platelet-derived growth factor (PDGFβ-r), it also decreases cell surface expression of major histocompatibility complex class I (MHCI) causing viral escape from immunosurveillance, and plays a role in the inhibition of the intracellular communication by means of aberrant connexin expression. E7 is considered as a weak transforming gene, it synergies with E5 in cell transformation during cancer development. E7 expression correlates in vivo with the over-expression of β1-integrin, which plays a role in the regulation of keratinocytes proliferation and differentiation. Additionally, E7 is involved in cell-mediated immune responses leading to tumour rejection, in anoikis process by direct binding to p600, and in invasion process by upregulation of Matrix metalloproteinase1 (MMP-1) expression. Studies on the role of BPV E5 and E7 oncoproteins in naturally occurring tumours are of scientific value, as they may shed new light on the biological role of these two oncogenes in cell transformation.

A case of urothelial carcinoma in situ of urinary bladder is reported in a 10-year-old cow naturally grazing on bracken-infested land. The cow suffered from enzootic hematuria for more than 5 years. The presence of bovine papillomavirus type 2 (BPV-2) DNA sequences was detected by polymerase chain reaction. The carcinoma in situ was characterized by the presence of anaplastic cells with amphophilic cytoplasm and pleomorphic nuclei containing granular, irregularly dispersed chromatin. Focal areas within the tumor contained large isolated and/or clustered cells. These cells had pale acidophilic cytoplasm, large nuclei with single or multiple nucleoli, and well-defined borders resembling Paget's cells. Immunohistochemically, all malignant cells were negative for vimentin and S-100 and positive for cytokeratins. In addition, normal and neoplastic cells expressed fragile histidine triad (FHIT) protein; surprisingly, some pagetoid cells did not. FHIT, the tumor suppressor gene at 3p14.2, encodes a protein of 147 amino acids (16.8 kd) with diadenosine triphosphate hydrolase activity and is a common target of deletions in human cancers of epithelial origin. Antibody to laminin detected a continuous epithelial basement membrane, thus clearly showing that neoplastic changes were limited to urothelial cells without invading stromal tissue. To our knowledge, this is the first report of an unusual pattern of spread of urothelial carcinoma in situ in a cow.

Several immunohistochemical markers have been used to define the differentiation pattern of urothelial cell tumors of the urinary bladder. We investigated the expression of the recently characterized uroplakin (UP) IIIb, an urothelium-specific and differentiation-dependent protein, in 39 urothelial tumors of the urinary bladder in cows that had suffered from chronic enzootic hematuria for several years. Bovine papillomavirus type 2 DNA was amplified and UP IIIb protein was detected in all these tumors. In papillomas and papillary carcinomas, UP IIIb expression was mostly seen as superficial staining; luminal and peripheral patterns were also observed. In nonpapillary carcinomas, UP IIIb appeared to define clearly the cell membrane lining intercellular and intracellular lumina as well as the cell borders in deeper cell layers. In benign and malignant lesions, an intracytoplasmic immunoreactivity was also detected. Coarse intracytoplasmic UP IIIb-positive material close to the nucleus occurred in some malignant cells. Focally strong membraneous immunostaining that marked single cells with complete ringlike peripheral pattern was seen. Although UP IIIb expression does not seem to correlate with the biological behavior of urothelial tumors, it appears to be a highly sensitive marker for bovine urothelial tumors.

Tuberculin skin test based on in vivo intradermal inoculation of purified protein derivative from Mycobacterium bovis (bPPD) is the diagnostic test for the control and surveillance of bovine tuberculosis (bTB). Proteomic analysis was performed on different bPPD preparations from M. bovis, strain AN5. Proteins were precipitated from bPPD solutions by TCA precipitation. The proteome of bPPD preparations was investigated by bottom-up proteomics, which consisted in protein digestion and nano-LC–MS/MS analysis. Mass spectrometry analysis was performed on a Q-exactive hybrid quadrupole-Orbitrap mass spectrometer coupled online to an Easy nano-LC1000 system. Three hundred and fifty-six proteins were identified and quantified by at least 2 peptides (99% confidence per peptide). One hundred and ninety-eight proteins, which had not been previously described, were detected; furthermore, the proteomic profile shared 80 proteins with previous proteomes from bPPDs from the United Kingdom and Brazil and 139 protein components from bPPD from Korea. Locus name of M. bovis (Mb) with orthologs from M. tuberculosis H37Rv, comparative gene and protein length, molecular mass, functional categories, gene name and function of each protein were reported. Ninety-two T cell mycobacterial antigens responsible for delayed-type hypersensitivity were detected, fifty-two of which were not previously reported in any bPPD proteome. Data are available via ProteomeXchange with identifier PXD005920. This study represents the highest proteome coverage of bPPD preparations to date. Since proteins perform cellular functions essential to health and/or disease, obtaining knowledge of their presence and variance is of great importance in understanding disease states and for advancing translational studies. Therefore, to better understand Mycobacterium tuberculosis complex biology during infection, survival, and persistence, the reproducible evaluation of the proteins that catalyze and control these processes is critically important. More active and more specific tuberculins would be desirable. Indeed, many antigens contained within bPPD are currently responsible for the cross-reactivity resulting in false-positive results as they are shared between non-tuberculous and tuberculous mycobacteria.

Background: Bovine papillomaviruses (BPVs) types 1 and 2 are the only known papillomaviruses able to jump the species. In fact, BPVs 1/2 induce neoplasia in their natural bovine host but infection is also associated to neoplastic skin lesions in equids termed sarcoids. The equine sarcoid is considered to be the most common equine cutaneous tumour worldwide for which no effective therapy is available. Very little is known about the molecular mechanisms underlying tumourigenesis, although genes contributing to sarcoid development have been identified. Several studies associate the development of cancer to the loss of function of a number of oncosuppressor genes. In this study the putative role of O⁶-methylguanine-DNA methyltrasferase (MGMT) was investigated for sarcoids. The expression of the oncosuppressor protein was assessed in normal and sarcoid cells and tissues. In addition, the DNA methylation profile was analysed to assess the role of epigenetic mechanism in regulation of MGMT expression. Results: A group of 15 equine sarcoids and two primary sarcoid cell lines (fibroblasts) were analyzed for the expression of MGMT protein by immunohistochemistry, immunofluorescence and Western blotting techniques. The sarcoid cell line EqSO4b and the tumour samples showed a reduction or absence of MGMT expression. To investigate the causes of deregulated MGMT expression, ten samples were analyzed for the DNA methylation profile of the CpG island associated to the MGMT promoter. The analysis of 73 CpGs encompassing the region of interest showed in 1 out of 10 (10%) sarcoids a pronouncedly altered methylation profile when compared to the control epidermal sample. Similarily the EqSO4b cell line showed an altered MGMT methylation pattern in comparison to normal fibroblasts. Conclusion: As previously demonstrated for the oncosuppressor gene FHIT, analysis of MGMT expression in sarcoid tissues and a sarcoid-derived fibroblast cell line further suggests that oncosuppressor silencing may be also involved in BPV-induced equine tumours. Abnormal DNA methylation seems to be one of the possible molecular mechanisms involved in the alteration of MGMT expression. Further studies are required to address other basic molecular mechanisms involved in reduced MGMT expression. This study underlines the possible role of DNA methylation in oncosuppressor inactivation in equine sarcoids.