Felis catus papillomavirus type-2 but not type-1 is detectable and transcriptionally active in the blood of healthy cats.

Papillomaviruses (PVs) are small DNA viruses that induce benign and/or malignant epithelial tumours in different species, including the domestic cat (Felis catus). To date, five F. catus papillomavirus genotypes have been identified (FcaPV-1 to FcaPV-5). FcaPV-1 is associated with skin and oral benign lesions, while FcaPV-2 infection is widely associated with feline squamous cell carcinomas. Several human and animal PVs have been found in body fluids such as peripheral blood; however, the presence of FcaPVs in non-epithelial tissues has not previously been investigated. The aim of this study was to assess the presence and gene expression of FcaPV-1 and FcaPV-2 in the blood of healthy cats. We detected FcaPV-2 DNA in 26 of 103 (25%) blood samples. Importantly, FcaPV-2 L1, E2, E6 and E7 genes were found to be expressed in 3 (25%), 11 (92%), 6 (50%) and 5 (42%) of the samples available for mRNA analysis, respectively. FcaPV-1 was not detected in any of the blood samples analysed here. The data obtained in this work suggest active and eventually productive infection of FcaPV-2 in the blood of healthy cats, implying a possible role in intra-individual spreading as well as in vertical and horizontal transmission.

Detection of Papillomavirus DNA in Cutaneous Squamous Cell Carcinoma and Multiple Papillomas in Captive Reptiles.

Papillomaviruses (PVs) are small, non-enveloped DNA viruses that cause mucocutaneous tumours including squamous cell carcinoma (SCC) in man. In animals, evidence supports a causal role for PVs in the development of cutaneous and oral SCC in some species. In reptiles, three cases of papilloma or fibropapilloma have been associated with PV infection, but no association has been reported to date with SCC. Two cases of cutaneous epithelial tumours, multiple papillomas in a spiny-tailed lizard (Uromastyx acanthinura) and SCC in a Dumeril's boa (Acrantophis dumerili), were investigated by polymerase chain reaction. PV DNA was amplified from samples of both lesions. Typical microscopical features suggestive of PV infection (e.g. the presence of koilocytes) were observed in the lesions from the spiny-tailed lizard. This is the first report of an association between PV and SCC in reptiles. Further studies are needed to better clarify the role of PVs in these species and to characterize the PV strains involved.

Bovine Papillomavirus: New Insights into an Old Disease.

Bovine papillomaviruses (BPVs) are small DNA tumoral viruses able to induce benign cutaneous and/or mucosal epithelial lesions. Generally, the benign tumours affecting the skin or mucosa spontaneously regress, but under special circumstances, the defence system may be overwhelmed, thus leading to cancer, especially in the presence of immunosuppressant and mutagen agents from bracken fern. To date, thirteen different BPV genotypes have been associated with skin and mucosal tumours in cattle, and out of these, only four types (BPV-1, -2, -5 and -13) cross-infect other species. Recent investigations in vivo have revealed new insights into the epidemiology and pathogenesis of this viral infection. This review briefly discusses viral epidemiology, will give data on BPV genome structure and viral genes and will describe the cellular events and new aspects of both cutaneous and mucosal tumours in large ruminants. Finally, some aspects of active immunization will be described.

Expression of Cyclooxygenase-2 in naturally occurring bovine cutaneous fibropapillomas.

Bovine cutaneous fibropapillomas are benign skin tumours characterized by epithelial and dermal proliferation and induced by Bovine papillomaviruses (BPVs). Cyclooxygenase (COX) 1 and 2 are enzymes involved in pathological conditions, such as inflammation and epithelial carcinogenesis. Here we investigated biochemically and immunohistochemically COX-2 expression in bovine cutaneous fibropapillomas. Eight of twelve fibropapillomas (67%) showed COX-2 positive immunosignal mostly in the cytoplasm of the basal cell layer, while the normal skin did not stain. Biochemical analysis confirmed the expression of COX-2 in tumour samples. This study shows COX-2 expression in cutaneous fibropapillomas, suggesting a contribution in epithelial tumour development.

The presence and gene expression of bovine papillomavirus in the peripheral blood and semen of healthy horses.

Papillomavirus (PV) are double-stranded DNA viruses that can cause both benignant and malignant tumours in mammals. Twelve genotypes of bovine papillomavirus (BPV1-12) have been identified so far. The presence of BPV1 and 2 has been found in the body fluids of cattle and horses. The aim of this study is to investigate the presence of BPV DNA and the expression of viral genes in the blood and sperm cells of healthy horses using PCR and RT-PCR. BPV-1 or 2 was detected in 14 of 70 blood samples (20%) and in 11 of 31 semen samples (35%). In five of fourteen blood samples, the E5 expression tested positive, while no blood sample was positive for L1 expression. Four of 11 (36%) semen cell samples proved to be positive for E5 expression, while no gene expression in L1 could be detected. This is the first study that shows BPV1 gene expression in the blood and semen of healthy horses. Our data illustrate the need for a better understanding of the presence of BPV in non-epithelial tissues of horses and their role in the vertical and horizontal transmission of these viruses.

Expression of platelet derived growth factor ß receptor, its activation and downstream signals in bovine cutaneous fibropapillomas.

Bovine cutaneous fibropapillomas are benign skin tumours formed by proliferation of epidermal keratinocytes and dermal fibroblasts caused by bovine papillomaviruses (BPVs). BPV E5 oncoprotein plays a key role in neoplastic cell transformation by specifically binding to the platelet derived growth factor beta receptor (PDGFßR) causing its phosphorylation and activation of proliferation and survival signal transduction pathways, among these phosphatidyl inositol-3-kinase (PI3K)/Akt and Ras-mitogen-activated-protein-kinase-Erk (Ras-MAPK-Erk) pathways. The aim of this study was to investigate the expression of PDGFßR, its phosphorylation status and expression of the downstream molecules phospho-Akt (pAkt) and phospho-Erk (pErk), in naturally occurring bovine cutaneous fibropapillomas. By immunohistochemistry on serial sections we showed cytoplasmic co-expression of the PDGFßR and E5 protein in neoplastic tissue. Western blot analysis revealed that PDGFßR was phosphorylated in higher amount in tumour samples compared to normal skin. pAkt, but not pErk, was also overexpressed in tumour samples. These findings may provide new insights into the aetiopathogenic mechanisms underlying naturally occurring bovine fibropapillomas and contribute to understanding the molecular scenario underlying BPV induced tumourigenesis.

Expression of gap junction protein connexin 43 in bovine urinary bladder tumours.

The aetiopathogenesis of urinary bladder tumours in cattle involves prolonged ingestion of bracken fern and infection by bovine papillomavirus types 1 or 2 (BPV-1/2). The oncogenic activity of BPV is largely associated with the major oncoprotein E5. Gap junctions are the only communicating junctions found in animal tissues and are composed of proteins known as connexins. Alterations in connexin expression have been associated with oncogenesis. The present study investigated biochemically and immunohistochemically the expression of connexin 43 in samples of normal (n=2), dysplastic (n=3) and neoplastic (n=23) bovine urothelium. The tumours included 10 carcinomas in situ, five papillary urothelial carcinomas and eight invasive urothelial carcinomas. Normal and dysplastic urothelium had membrane expression of connexin 43, but this was reduced in samples of carcinoma in situ. Papillary urothelial carcinomas showed moderate cytoplasmic and membrane labelling, while invasive carcinoma showed loss of connexin 43 expression.

Phosphatidylinositol-3-kinase-AKT pathway, phospho-JUN and phospho-JNK expression in spontaneously arising bovine urinary bladder tumours.

The aetiopathogenesis of urinary bladder tumours in cattle involves prolonged ingestion of bracken fern and infection by bovine papillomavirus types 1 or 2 (BPV-1/2). E5, the major BPV-1/2 oncoprotein, binds to the activated platelet-derived growth factor beta receptor (pPDGF-betaR), inducing cell transformation in vitro and spontaneously arising urinary bladder tumours. The aim of this study was to assess whether the 85 kDa regulatory subunit (p85) of the phosphatidylinositol-3-kinase (PI3K)-AKT pathway and other transforming signals phospho-JUN (pJUN) and phospho-JUN N-terminal kinases (pJNK) may be important in the development of BPV-associated urothelial carcinomas. A physical interaction between the pPDGF-betaR and PI3K was shown in four tumours and two samples of normal bladder tissue by co-immunoprecipitation and western blotting. There was greater expression of the PI3K-AKT-cyclin D3 molecular pathway downstream to the activation of pPDGF-betaR in neoplastic compared with normal tissue. pJNK and pJUN were overexpressed in samples of tumour compared with normal mucosal tissue. These findings provide new insights into the aetiopathogenic mechanisms underlying naturally occurring bovine urothelial carcinogenesis and contribute to understanding of the role of E5 oncoprotein in naturally occurring tumorigenesis.

Expression of endothelin-1 and endothelin receptor a in canine ovarian tumours.

Ovarian tumours have a low incidence in bitch. Endothelin (ET-1) and endothelin A receptor (ET-A) are overexpressed in human ovarian cancer. Twenty canine ovarian tumours and five normal samples were first evaluated by western blotting and then immunohistochemically for ET-1 and ET-A expression. Seventeen out of twenty tumours were ET-1 positive. Eight out of twenty tumours were ET-A immunohistochemically positive. At molecular level both proteins were proven to be expressed in normal as well as in tumour samples. Our results show that ET-1 and ET-A are overexpressed in canine ovarian tumours, suggesting a potential role of these two molecules in canine ovarian carcinogenesis.

Cryopreservation of feline epididymal spermatozoa from dead and alive animals and its use in assisted reproduction.

Cryopreservation of gametes is an important tool in assisted reproduction programmes; long-term storage of oocytes or spermatozoa is necessary when in vitro fertilization (IVF) or artificial insemination is to be performed at a future date. Cryopreservation of epididymal spermatozoa offers a potential tool for rescuing genetic material from males of endangered populations. The objectives of this work were to: (1) examine sperm motility, viability, abnormality and acrosome integrity of frozen-thawed domestic cat epididymal spermatozoa; and (2) evaluate the same cryopreservation method on wild feline spermatozoa, needed to preserve their genetic resources. Epididymides were collected from 20 domestic cats during routine neutering procedure and from two wild felines at autopsy. The sperm samples, diluted with 4% glycerol/Tris/egg yolk, were loaded into 0.25 ml mini-straws, exposed to nitrogen vapour and stored in liquid nitrogen. After 4 weeks, samples were thawed and re-evaluated. The quality of each fresh and frozen-thawed sperm sample was tested by determining the motility (54.7 +/- 11.3% and 32 +/- 13.1% respectively for cat spermatozoa; 38.3 +/- 18.7% and 21.5 +/- 16.8% respectively for tiger spermatozoa), viability (74.3 +/- 8.6% and 45.2 +/- 9.4% respectively for cat spermatozoa; 42.4 +/- 14.5% and 33.5 +/- 12.9% respectively for wild felid spermatozoa), morphology and acrosomal status. The present study showed that feline epididymal spermatozoa can be frozen in egg-yolk extender with 4.0% glycerol in 0.25 ml straws. The procedure used in the present study for epididymal cat sperm cryopreservation may be applied to bank the genetic resources of wild felid species.

A review of bovine urothelial tumours and tumour-like lesions of the urinary bladder.

Four hundred bovine urothelial tumours and tumour-like lesions were classified in accordance with the 2004 World Health Organization (WHO) morphological classification for human urothelial tumours. The spectrum of neoplastic lesions of the urinary bladder of cattle is becoming wider and bovine urothelial tumours share striking morphological features with their human counterparts. A classification system based on the WHO scheme would also be appropriate for the classification of bovine bladder tumours. Bovine urothelial tumours are most often multiple. Four distinct growth patterns of bovine urothelial tumours and tumour-like lesions are recognized: flat, exophytic or papillary, endophytic and invasive. Carcinoma in situ (CIS) is the most common flat urothelial lesion, accounting for approximately 4% of urothelial tumours. CIS is detected adjacent to papillary and invasive tumours in 80-90% of cases. Approximately 3% of papillary lesions are papillomas and approximately 5% are 'papillary urothelial neoplasms of low malignant potential' (PUNLMP). Low-grade carcinoma is the most common urothelial tumour of cattle. High-grade carcinomas, and low and high-grade invasive tumours, are less commonly seen. Bovine papillomavirus (BPV) infection and ingestion of bracken fern both play a central role in carcinogenesis of these lesions.

Ferritin heavy chain (FHC) is up-regulated in papillomavirus-associated urothelial tumours of the urinary bladder in cattle.

The up-regulation of ferritin heavy chain (FHC) is reported in six papillary and in four invasive urothelial tumours of the urinary bladder of cattle grazing on mountain pastures rich in bracken fern. All tumours contained sequence of bovine papillomavirus type-2 (BPV-2) as determined by polymerase chain reaction (PCR) analyses and validated by direct sequencing of the amplified products. The oncoprotein E5 was also detected in these tumours by immunoprecipitation and by immunofluorescence and laser scanning confocal microscopy. Expression of FHC was evaluated by western blot analysis, reverse transcriptase (RT) PCR, real-time RT-PCR and immunohistochemistry. The oligonucleotide sequence of the bovine ferritin amplicons was identical to that of human ferritin. Nuclear overexpression of p65, an important component of nuclear factor kappaB (NF-kappaB) transcription factors, was also observed. These findings suggest that FHC up-regulation may be mediated by activation of NF-kappaB and that in turn this may be related to the resistance of bovine papillomavirus type-2 (BPV-2) infected urothelial cells to apoptosis.

Co-expression of bovine papillomavirus E5 and E7 oncoproteins in naturally occurring carcinomas of the urinary bladder in cattle.

The aetiopathogenesis of urinary bladder tumours in cattle involves prolonged ingestion of bracken fern and infection by bovine papillomavirus (BPV). The aim of the present study was to determine whether there was co-expression of BPV oncoproteins E5 and E7 in such urothelial carcinomas. Fifteen samples were shown by immunohistochemistry to express E7 with labelling of both the cytoplasm and nucleus, in addition to labelling of the urothelial cell membrane. Three of these samples were subsequently investigated by dual-labelling immunofluorescence and co-expression of E5 and E7 was demonstrated. This is the first report of co-expression of these two oncoproteins in bovine urinary bladder carcinomas. The results suggest that the E7 oncogene has a role in urothelial carcinogenesis.

Bovine papillomavirus type 1 DNA and E5 oncoprotein expression in water buffalo fibropapillomas.

Papillomas and fibropapillomas may occur in the skin and in different organs in animals. Ten different genotypes of bovine papillomavirus (BPV) have been identified. BPV-1 through BPV-10 are all strictly species-specific, but BPV-1/2 may also infect other species such as equids, inducing fibroblastic tumors. BPV-1 and BPV-2 are associated with fibropapillomas in cattle; these tumors are formed by excessive proliferation of virus-infected dermal fibroblasts and epidermal keratinocytes. Nine water buffalo (Bubalus bubalis) were examined for the presence of multiple cutaneous and perivulvar tumors. Cutaneous and perivulvar fibropapillomatosis were confirmed histologically. Negative-stain transmission electron microscopic examination revealed papillomavirus-like particles in the fibropapillomas, and papillomaviral DNA was also detected by the polymerase chain reaction. The amplified long control region (LCR) DNA sequence was identical to that of BPV-1. The BPV-1 E5 oncoprotein was strongly expressed in the tumor cells thus confirming a causal role of the virus. This article represents the first report of cutaneous, perivulvar, and vulvar fibropapilloma associated with BPV-1 infection in the water buffalo and describes another example of cross-species infection by BPV-1.

Activated platelet-derived growth factor beta receptor expression, PI3K-AKT pathway molecular analysis, and transforming signals in equine sarcoids.

The equine sarcoid is the most common dermatologic neoplasm reported in horses. Bovine papillomavirus (BPV) types 1 and 2 are associated with sarcoids, in which the expression of the major transforming oncoprotein (E5) is often recorded. The transformation activity of the virus is due to the binding of the E5 to the platelet-derived growth factor beta receptor (PDGFbeta-r). In the present study, we show by Western blot in 4 sarcoid samples and 3 normal equine skin samples that the PDGFbeta-r is more phosphorylated in sarcoid tissue than in normal skin (P < .001). Furthermore, the physical interaction between the activated receptor and the 85-kDa regulatory subunit (p85) of phosphatidylinositol-3-kinase (PI3K) is shown by coimmunoprecipitation. The PI3K-AKT-cyclin D3 molecular pathway downstream to the activation of the PDGFbeta-r is shown to be expressed, and the amount of the investigated molecules is higher than normal (P < .001), suggesting an activation of these effectors in sarcoids. Further, we demonstrate that phospho-JNK and phospho-JUN are more expressed in sarcoids than in normal skin. Our results provide new insights into the pathogenesis of equine sarcoids and support the validity of this in-vivo model to further characterize the molecular pathways underlying BPV E5-induced carcinogenesis.

Bilateral ovarian malignant mixed Mullerian tumor in a dog.

Malignant mixed Müllerian tumor (MMMT) is a rare neoplasm of the female genital tract. We report a case of bilateral ovarian MMMT in a 10-year-old female dog. Ovaries were only moderately enlarged with a papillary surface and firm nodules. Multiple metastases were observed in the abdominal cavity and pulmonary parenchyma. Histologically, both ovaries had intermingled carcinomatous and sarcomatous components with cartilage and bone. Metastatic lesions were not mixed. The peritoneal metastases were carcinomatous; pulmonary metastases were sarcomatous. Carcinomatous elements of the MMMT were immunohistochemically positive for cytokeratin (CK) AE1/AE3, CK7, and vimentin and estrogen receptors. Conversely, the sarcomatous cells were positive for vimentin, but negative for CKs. Chondrocytes also expressed S-100 protein. On the basis of similarities to human ovarian MMMT, the diagnosis was heterologous malignant mixed Müllerian tumor of the ovary.

Expression of platelet-derived growth factor-beta receptor and bovine papillomavirus E5 and E7 oncoproteins in equine sarcoid.

Equine sarcoids are benign fibroblastic skin tumours that are recognized throughout the world. Infection with bovine papillomavirus (BPV) types 1 and 2 has been implicated as a major factor in disease development; however, the cellular mechanisms underlying fibroblast transformation remain poorly defined. The present study further characterizes aspects of the association with BPV in 15 equine sarcoids. BPV DNA was demonstrated in 12/15 tumours collected from different areas of Italy. Nine of these 12 tumours expressed the BPV oncoproteins E5 and E7, but these oncoproteins were not expressed by normal equine cells. The BPV E5 protein is known to bind to the platelet-derived growth factor-beta receptor (PDGF-betaR) and this molecule was expressed by 11 of the 12 sarcoids in which E5 was demonstrated. These findings add further weight to the theory that BPV and the PDGF-betaR may have a role in the pathogenesis of this disease.

Lymphoepithelioma-like carcinoma of the urinary bladder in a cow associated with bovine papillomavirus type-2.

Lymphoepithelioma-like carcinoma (LELCA) of the urinary bladder is reported in a 7-year-old cow that had grazed pasture rich in bracken fern and had suffered from severe intermittent haematuria from 3 to 4 years of age. On necropsy examination there were multiple haemorrhagic foci scattered over the mucosal surface of the urinary bladder. Microscopically there were nests, cords and sheets of neoplastic cells infiltrating the lamina propria and muscularis propria. These had a syncytial appearance with ill-defined cytoplasmic borders, large nuclei and prominent nucleoli. There was a prominent associated inflammatory infiltrate comprising lymphocytes and plasma cells with sparse histiocytes and granulocytes. Immunohistochemically, LELCA cells expressed cytokeratin but not vimentin. The LELCA was focally admixed with a concomitant papillary high-grade carcinoma that also infiltrated the lamina propria. A diffuse carcinoma in situ was also present. Bovine papillomavirus type-2 (BPV-2) DNA was amplified from frozen neoplastic tissue and from selected areas of formalin-fixed, paraffin wax-embedded tissue obtained by laser capture microdissection. Microbiological culture of a urine sample resulted in isolation of Weeksella virosa, Rhizobium radiobacter and Staphylococcus warneri. Flow cytometric analysis performed on blood mononuclear cells revealed down-regulation of a panel of markers including CD3, CD4, CD8alpha, CD45, MHC class I and MHC class II (HLA-DRalpha, HLA-DQ, HLA-DP). This report extends the spectrum of neoplastic urothelial lesions described in cattle and provides further evidence that some features of these tumours are similar to human counterparts.

Multiple glomus tumors of the urinary bladder in a cow associated with bovine papillomavirus type 2 (BPV-2) infection.

We report a case of multiple glomus tumors associated with bovine papillomavirus type 2 (BPV-2) infection in the urinary bladder of a 13-year-old cow suffering from severe chronic enzootic hematuria. Macroscopically, multiple submucosal reddish nodules were seen swelling the vesical mucosa. Histologically, neoplastic proliferation was characterized by the presence of numerous blood vessels. These were lined by normal endothelial cells surrounded by round epithelioid cells with central nuclei, prominent nucleoli, acidophilic cytoplasm, and well-defined cytoplasmic borders. Tumor cells were distributed around open vascular lumina and in perivascular spaces. They were immunohistochemically positive for actin and vimentin and negative for cytokeratins, desmin, and factor VIII-related antigen. On the basis of these findings, this tumor was diagnosed as glomus tumor, a neoplasm not previously reported in cattle and exceedingly rare in animals. BPV-2 DNA was amplified from the formalin-fixed, paraffin-processed tissue specimens obtained by laser capture microdissection. This report widens the spectrum of mesenchymal tumors of the bovine urinary bladder. Finally, the microscopic pattern of tumor described here shares striking morphologic and immunohistochemical similarities with the angiomatous form of glomus tumor known to occur in man.