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Bovine Papillomaviruses (BPVs) constitute a diverse group within the Papillomaviridae family, playing a crucial role in bovine health and economic considerations. This study investigates the dynamics of vertical transmission of BPV in cattle, focusing on five cows and their reproductive tissues, as well as three gravid cows and their fetuses. DNA and RNA samples were extracted from the warts, fetal skin, placenta, uterus, ovary, and blood of cows, as well as the skin and blood of fetuses. Polymerase Chain Reaction (PCR) targeted BPV types 1-6 and 8-14, was assessed in both cows and fetuses. Additionally, Reverse Transcription PCR (RT-PCR) examined BPV-2 E5 oncogene expression in the skin and reproductive sites of mother cows and fetuses. Our findings unveil a rich diversity of BPV types, including BPV-2, 3, 9, 10, 12, and 14, present in both maternal and fetal tissues. Intriguingly, certain types, namely BPV-4, 6, 8, and 11, were exclusively identified in maternal tissues A higher diversity of BPVs was observed in cow warts, followed by cow blood, fetal blood, and fetal skin. Strikingly similar BPV types in gravid cow blood and fetuses suggest primary dissemination through the bloodstream and transmission via the placenta, though detected in lower numbers in cow uterus and ovary. Histopathological analysis revealed no abnormalities in the reproductive tissues despite the presence of BPV. However, in one bladder sample from a cow that did not consume bracken fern, urothelial neoplasia in situ was observed. The study extends beyond detection, exploring the expression of the BPV-2 E5 oncogene in fetal tissues, providing insights into potential cell implications. Comparative analyses with previous studies highlight the uniqueness of our investigation, encompassing a broader array of BPV types in the gravid cows and their fetuses. The findings not only establish a foundation for further investigations into the mechanisms of vertical transmission but also highlight the need for targeted interventions and surveillance strategies to mitigate potential health risks associated with specific BPV types., (© 2024. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

Bovine papillomatosis is an infectious viral disease of cattle characterized by development of benign cutaneous warts. The present study describes bovine papillomavirus infection in cattle on clinco-pathological and molecular bases and compares the identified strains with the previously characterized papillomavirus isolates in Egypt either of bovine or equine origin. Out of sixty examined cattle, skin lesions were collected from eleven clinically diseased cattle exhibiting typical papillomatosis clinical signs and subjected to histopathological and molecular identification. Histological sections showed well-developed papillary projections of squamous epithelium associated with fibrovascular stroma. Type 1 bovine papillomavirus (BPV-1) was identified in the cutaneous lesions based on the results of L1 gene-based PCR using degenerated primer followed by DNA sequencing. Comparative sequence and evolutionary analysis revealed that papilloma sequences (OP777901, OP777902, OP777903) obtained in the current study are clustered along with MW018705.1, MG547343.1isolated from cattle in Egypt in 2017/2018 and MT502095.1.1, and MT502105.1 isolated from equine in Egypt in 2019. Results prove the circulation of BPV-1 in the areas under investigation and shed light on the role of multispecies grazing in Egypt as a risk factor for transmission of BPV-1 from cattle to horses.

In this study, the digital droplet polymerase chain reaction (ddPCR) was used to quantify circulating bovine papillomavirus (BPV; genus: Deltapapillomavirus) DNA levels in blood samples from 25 clinically normal cows and 15 cows with chronic enzootic haematuria due to papillomavirus-associated bladder tumours. ddPCR detected BPV DNA in 95% of all the samples (i.e. in 24 of the clinically normal cows and 14 of the diseased animals), whereas quantitative real-time PCR (qPCR) detected it in only 57.5% of the same blood samples, with percentage differences between ddPCR and qPCR being statistically significant (p-value ≤ .05), according to chi-squared test. Furthermore, ddPCR detected BPV infections by a single genotype and by multiple genotypes in 37% and 63% of the cows, whereas qPCR detected these in 16% and 16%. Of the two assays, ddPCR was the more sensitive and accurate clinical diagnostic tool, allowing the detection of otherwise undetectable BPV genotypes, and consequently, a higher number of BPV co-infections. qPCR failed to detect many BPV co-infections by multiple genotypes. Therefore, ddPCR may be an essential tool for improving diagnostic procedures, allowing the identification of the genotypic distribution of BPV and a better understanding about the territorial divergence, if any, of the BPV prevalence in different areas. No significant differences in the blood viral load estimations were observed between the two animal groups, suggesting that the bloodstream could be a site of primary infection. Finally, as BPV DNA was detected in cows affected by non-invasive urothelial tumours, including papilloma and papillary urothelial neoplasms of low malignant potential, the circulating BPVs appeared to be independent of the status of urothelial neoplasms. Therefore, unlike in humans, circulating BPVs cannot be an actual prognostic marker of urothelial tumours in cows., (© 2020 Wiley-VCH GmbH.)

Equine sarcoids (ES) were diagnosed in 12 Somali wild asses (SWA) ( Equus africanus somaliensis ) from 10 different institutions of the SWA European Endangered Species Programme from 1976 to 2019. Samples of surgically excised masses, biopsies, or necropsy samples were submitted for histologic and virologic analysis. In addition, tissue samples from one onager ( Equus hemionus onager ), one kulan ( Equus hemionus kulan ), and two Hartmann's mountain zebras (HMZ) ( Equus zebra hartmannae ) were examined. Histology confirmed the diagnosis of ES exhibiting the typical microscopic features. Polymerase chain reaction detected bovine papillomavirus type 1 (BPV1) DNA in eight SWA samples and bovine papillomavirus type 2 (BPV2) DNA in one SWA sample. The onager, kulan, and one HMZ sample tested positive for BPV1. The other HMZ tested positive for BPV1 and BPV2. This is the first report of ES in an onager. Surgical excision was the treatment elected by most veterinarians. A follow-up survey of the cases over several years after clinical diagnosis and therapy revealed variable individual outcome with ES recurrence in four cases. Three SWA and the kulan were euthanized due to the severity of the lesions. Nine affected SWA were males with seven having a sarcoid located at the prepuce. Because a genetic disposition is a risk factor for the development of ES in horses, this may also be true for endangered wild equids with few founder animals in their studbook history. Innovative approaches regarding therapy and prevention of ES in wild equids are therefore highly encouraged.

Impaired keratinocyte differentiation has recently been suggested as a key event in equine hoof canker development. Koilocytotic appearance of keratinocytes, one of the most characteristic morphological alterations in hoof canker tissue, is also a common marker for papillomavirus (PV) infection, and bovine PV-1 and/or -2 (BPV-1/2) has previously been detected in equine canker patients. Therefore, the present study aimed to correlate the frequency and severity of koilocytotic keratinocytes with BPV detection in hoof canker samples. Hoof tissue of 5/18 canker-affected horses and 2/6 control horses tested positive for BPV-1/2 DNA using polymerase chain reaction. Thus, no association between the presence of BPV-1/2 papillomaviral DNA and koilocytotic appearance was found. Proteins associated with but not specific for PV infection were also investigated. Using immunohistochemistry, specific adhesion molecules (E-cadherin and β-catenin) and intermediate filaments (keratins 6 and 14) important for intact epidermal barrier function and keratinocyte differentiation were documented in control samples ( n = 6) and in hoof canker tissue samples ( n = 19). Altered expression patterns of intermediate filaments and adhesion molecules were demonstrated in canker tissue, confirming the importance of incomplete keratinocyte differentiation, as well as the crucial role of keratinocyte differentiation in hoof canker.

Thirty-one bovine cutaneous warts were submitted to macroscopic and histological analyses and to molecular analyses to partial amplification and sequencing of the L1 gene of bovine papillomavirus (BPV). Viral types detected were BPV1 (52%), BPV2 (29%), BPV6 (16%) and BPV10 (3%). BPV2 had lower frequency in papilloma in comparison to that in fibropapilloma (p = 0.002).

Background: There is a large body of evidence supporting bovine papillomavirus types 1 and 2 (BPV1; BPV2) as aetiological agents of equine sarcoids. However, there is conflicting data regarding BPV1/2 infection in sarcoid-free equids., Objectives: Data obtained between 2007 and 2017 by BPV1/2 screening of sarcoids and nonsarcoid tumours vs. samples from healthy equids are presented to help clarify this issue., Study Design: Cross-sectional study., Methods: Tumour material obtained from horses, donkeys and mules with confirmed sarcoids (n = 130), suspected sarcoids (n = 120), or nonsarcoid lesions (n = 70), skin biopsies from 102 tumour-free horses and dandruff/hair roots from 35 tumour-free donkeys and mules were screened for BPV1/2 infection. Sample DNA was extracted and validated by equine β-actin PCR. BPV1/2 screening was performed by BPV1/2 E5-specific PCR allowing for the detection of less than 10 viral DNA molecules. Twenty-six amplicons were bidirectionally sequenced and compared to known E5 variants using BLAST program., Results: BPV1/2 E5 PCR scored positive for 130/130 diagnosed sarcoids, 63/120 suspected sarcoids and 13/70 nonsarcoid lesions, whereas 137/137 DNA aliquots derived from tumour-free equids tested negative. On predicted E5 protein level, six different BPV1 E5 variants were identified., Main Limitations: The diagnosis of equine sarcoid was not confirmed in 120 lesions., Conclusions: Lack of BPV1/2 E5 DNA in tumour-free equids and the prevalence of sarcoid disease in young adult individuals suggest that the time span between initial infection and sarcoid development is short. This contrasts with the long phase of virus latency characterising infection of humans by carcinogenic papillomaviruses. Presence of BPV1/2 DNA in several cases of poor wound healing/hypergranulation and dermatitis points to these skin disorders being possibly co-induced by BPV1/2. PCR screening of tumour tissue/scrapings for BPV1/2 DNA represents a reliable tool for the rapid validation of a clinical diagnosis of equine sarcoid., (© 2018 EVJ Ltd.)

Papillomavirus (PV) is a well-known pathogen associated with epithelial and mucosal neoplastic diseases. In contrast to human PVs, characterization of animal PVs from the aspect of anogenital neoplasm is still on a learning curve. In the present study, two vulval and one anal warts, histologically diagnosed as fibropapillomas, excised from dairy cattle were analyzed. PCR and sequencing revealed that bovine papillomavirus type 1 (BPV-1) and BPV-2 were detected from anal and vulval fibropapillomas, respectively. Immunohistochemistry detected PV antigen in a few differentiated keratinocytes of one vulval case. Reverse-transcriptase PCR detected the early region, but not the late region of BPV mRNA in all three cases. The present study will provide new insight into the relationship between BPV and anogenital papilloma in cattle.

To date, there have been no reports of coinfection with bovine papular stomatitis virus (BPSV) and bovine papillomavirus (BPV) in the same lesion. In the present study, one lingual papilloma-like sample was collected at an abattoir from the tongue of a 31-month-old Japanese black cow. Coinfection with BPSV and BPV was confirmed by histopathology, immunohistochemistry, PCR and RT-PCR. The evidence for coinfection with BPSV and BPV in the same lesion and an association of BPV with lingual papillomatosis will contribute to future epidemiological studies of these two viruses.

Background: Equine sarcoids are the most common neoplasms in horses. Bovine papilloma- virus type 1 (BPV-1) is the main viral type identified in equine sarcoids in Europe., Objective: The aim of the present study was to genetically evaluate BPV types based on DNA analyses of the CDS of the L1 gene. The presence of BPV DNA was confirmed by Degenerate Oligonucleotide-Primed Polymerase Chain Reaction (DOP PCR) with FAP59/FAP64 consensus primers., Results: The DNA was detected in 21/40 (52.5%) of clinically diagnosed sarcoids. More than half of 14 isolates (66.7%) shared 100% homology with BPV-1 Deltapapillomavirus 4 isolate 09 asi UK (Acc. No. MF384289) and 99% nucleotide identity with BPV-1 isolate EqSarc1 (Acc. No. JX678969). A comparison with BPV-1 isolate EqSarc1 revealed one silent mutation in C5827T which did not change the aminoacid codon. The remaining 6 isolates (28.6%) shared 100% nucleotide identity with the BPV-1 (Acc. No. X02346) "wild type" isolate, and 1 isolate (4.8%) demonstrated 99% nucleotide identity with BPV-2 (Acc. No. M20219)., Conclusions: Variants of BPV-1 isolate EqSarc1 (Acc. No. JX678969) constitute the most prevalent type of BPV-1 in Polish horses., (Copyright© by the Polish Academy of Sciences.)

Papillomaviruses are among the most widespread animal viruses, with many hosts harbouring multiple virus types. The present study aimed to evaluate the haematological and immunophenotypic profile of cattle infected with bovine papillomavirus (BPV). Blood samples were collected from 10 animals with clinical cutaneous BPV and without clinical papillomatosis (control). Haematological analysis demonstrated a significant reduction in haemoglobin and haematocrit for BPV-infected animals. The results also showed an increase of natural killer cells and a decrease of γδ + T-cells and the CD4 + /CD8 + ratio for the BPV group when compared to the control group. The infection was also found to stimulate a pro-inflammatory profile with the participation of CD8 + T cells producing elevated IFN-γ and IL-17. These findings, although preliminary, provide a better understanding of the immune response of cattle infected with BPV., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Bovine papillomavirus (BPV) belongs to the Papillomaviridae family and infects epithelial cells of bovines and closely related animals, causing hyperproliferative lesions known as warts or papillomas, which may regress or progress to form benign or malignant tumors. The virus enters the host cell and interacts with it by altering the regulation of genes that are responsible for controlling the cell cycle, thus triggering lesion formation. It is not yet known which host genes are regulated by viral infection. Therefore, the objective of this study was to make use of next-generation RNA sequencing methods to identify differentially expressed genes associated with BPV infection, which might elucidate possible marker genes that could be used to control the disease., Results: Transcriptome analysis revealed that 1343 genes were differentially regulated (FDR < 0.05). A comparison of gene expression in infected and noninfected cows indicated that 655 genes were significantly upregulated, and 688 genes were significantly downregulated. Most differentially expressed genes were associated with BPV infection pathways, which supports the hypothesis that viral infection was the mechanism associated with this regulation., Conclusions: This is the first study that focused on a large-scale evaluation of gene expression associated with BPV infection, which is important to identify possible metabolic pathways regulated by host genes for lesion development. In addition, novel targets could be identified in order to find ligands that interact with BPV, with the aim of interrupting the infection cycle.

Severe papillomatosis occasionally causes astasia leading to euthanizing cattle. There are currently a limited number of reports on virologic approach in severe bovine papillomatosis. Here we report a full genome characterization of bovine papillomavirus type 1 (BPV-1) from the case of severe papillomatosis. A calf developed numerous papillomas on the skin and some nodules in the upper gastrointestinal tract at seven months old. The skin lesion was diagnosed as the epithelial papilloma with BPV antigen expression, while the gastrointestinal lesions were diagnosed as the fibropapilloma without BPV antigen. Full genome analysis revealed that BPV-1s detected in all the lesions were exactly the same. Compared with the reference BPV-1 sequence, there was a single nucleotide insertion in the upstream regulatory region.

An 8-month-old male Japanese Black calf was referred for the evaluation of a slow-growing conjunctival mass in the right eye. A superficial keratectomy was performed followed by recurrence on two occasions. No metastases were found in surrounding tissues. Histological, immunohistochemical, and ultrastructural investigation revealed that both the primary and the recurrent lesions were benign, conjunctival, myofibroblastomas. Interestingly, bovine papillomavirus type 2 (BPV-2) DNA was detected in both myofibroblastoma lesions. Archival bovine myofibroblastomas from the vulva and neck were also analyzed for papillomaviral genomes. BPV-2 DNA was also amplified from these lesions. To the best of our knowledge, this is the first report describing a potential causal relationship between BPV-2 infection and conjunctival myofibroblastoma.

CASE HISTORY Anal warts were observed in heifers in two unrelated groups of animals. Heifers in one group developed visible warts 4 months after manual rectal examination and heifers in the other group developed warts 5 months after examination using a hand-held rectal ultrasound probe. CLINICAL FINDINGS Large exophytic proliferative anal masses were observed in 5/15 (33%) heifers in one group and 13/149 (9%) heifers in the second group. Heifers in the second group were also noted to have similar masses on the underside of the tail at sites previously used for venepuncture and some of the heifers had skin warts. Despite the large size of the anal masses, none of the heifers showed clinical signs of systemic illness. HISTOPATHOLOGICAL FINDINGS An anal mass was removed from one heifer in each of the two groups. Sections from both masses showed hyperplastic epithelium covering a proliferation of well-differentiated fibroblasts consistent with fibropapillomas. Small numbers of cells within the epidermis had clear cytoplasm with clumped keratohyalin granules. MOLECULAR BIOLOGY Bovine papillomavirus (BPV) type 2 DNA was amplified from both fibropapillomas by PCR. DIAGNOSIS Multiple anal fibropapillomas associated with BPV-2. CLINICAL RELEVANCE Bovine anal fibropapillomas have only been reported in heifers that have undergone rectal examination, and infection of anal microabrasions in an immunologically naïve animal appears to be associated with disease development. The source and method of spread of BPV-2 within these groups could not be determined. However spread of BPV-2 within the groups by the veterinarian performing rectal examinations may have been most likely. While these fibropapillomas had a dramatic appearance, like fibropapillomas elsewhere on the body, they did not have any significant effect on the health of the affected heifers. As these lesions can be diagnosed by clinical examination and self-resolve without treatment, it is important that veterinarians are aware of this rare manifestation of papillomavirus infection of cattle.

This study diagnosed cutaneous wart lesions excised from three rams from a sheep farm in São Paulo State, Brazil. Histopathologically, these cases were diagnosed as papilloma. The amplification by PCR, sequencing and bioinformatics analysis showed that all the lesions presented DNA sequences of bovine papillomavirus type 2. This is the first report confirming the detection of BPV2 in papilloma warts from ovines., (© 2018 Blackwell Verlag GmbH.)

Several attempts have been made to categorize equid- and bovid-specific bovine papillomavirus 1 (BPV1) isolates based on sequence tags. This study includes newly determined sequence information from 33 BPV1 isolates of equine, asinine and bovine origin and investigates sequence bias due to host species. Twenty of the viral genomes were sequenced over their entire length and a further thirteen were sequenced, including flanking sequences, at two specific sites, the LCR and the E5 ORF. Alignment and analyses of the sequences did not reveal statistically significant site differences between the sequences of bovine and equid origin. None of the proposed sites of divergence noted by other authors demonstrated significant species-specific characteristics. Our results suggest that BPV1 is shared between equine, asinine and bovine host species, and that viral transfer between bovines and equids is a repeated and ongoing phenomenon., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The association between bovine papillomavirus (BPV) and equine sarcoids is well established, but it is unclear how the virus spreads. Although evidence in support of viral spread through direct animal contact exists, this does not explain sarcoid development in isolated equids. BPV DNA has been detected in flies, which could indicate that these insects serve as a vector. This study aimed to investigate whether BPV-negative stable flies (Stomoxys calcitrans) become positive for BPV DNA after exposure to equine sarcoid or bovine papilloma tissue under experimental conditions and, if so, for how long. A total of 420 stable flies were caught alive and exposed to BPV positive equine sarcoid or bovine papilloma tissue. During the following week, dead flies were collected daily and BPV loads were determined by quantitative PCR. There was a significant rise in BPV load after tissue exposure both in sarcoid and papilloma exposed flies, but the viral load was higher and remained high for a longer time after exposure to papilloma tissue compared to sarcoid tissue. Within days, viral loads decreased again and became indifferent from loads before exposure. The results of these experiments indicate that BPV transmission by S. calcitrans seems possible and is more likely to occur after contact with bovine papillomas than with equine sarcoids. Transmission seems only possible shortly after tissue exposure. Further research could include experimental induction of sarcoids with BPV positive stable flies, or a repeat of the experiment with micro-dissection prior to PCR., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Papillomaviruses (PVs) are epitheliotropic viruses that cause benign proliferative lesions in the skin (warts or papillomas) and mucous membranes of their natural hosts. In bovines specifically, 13 types of Bovine papillomaviruses (BPVs) are currently described in the literature, although the actual number may be greater than 20. BPV types are classified into four genera based on homology within the genomic regions of the L1 ORF, the most conserved sequence. This study conducted molecular typing of BPV in dairy cows with different papillomatosis cases and investigated the presence of co-infections across distinct BPV types in the same sample. After carrying out PCR using degenerate primers and type specific primers, 35 BPV suspected samples were detected as positive for BPV and these samples were used for typing using sequence analysis/PCR with type-specific primers. This analysis identified BPV-1, -2, -3, -4, -6, -7, -9 and -10, new putative types (BPV/BR/UEL6-like viruses) and the previously described putative type viruses (BAPV-6) in the 35 BPV-positive samples. In addition, co-infections across different BPV types were widely detected in the BPV-positive samples.  This study shows that PCR assays using degenerate primers to amplify partial fragments of the L1 gene followed by sequencing is useful for genotyping BPV. However, results need confirmation using type-specific primers in order to consider co-infections. In addition, this study identified a new putative type (in the same cluster as BPV/BR/UEL6-like viruses) and the previously described putative type viruses (BAPV-6) in teat papillomatosis of Turkish dairy cows. The study shows that it is essential to identify BPV types and their prevalence/distribution, and also to determine the clinical consequences of infection for the development of prophylactic and/or therapeutic procedures.

We have previously shown that immunization of horses with bovine papillomavirus type 1 (BPV1) L1 virus-like particles (VLPs) is safe and highly immunogenic and that BPV1 and bovine papillomavirus type 2 (BPV2) are closely related serotypes. Here we evaluated the protective potential of a BPV1 L1 VLP vaccine against experimental BPV1 and BPV2 challenge and studied the safety and immunogenicity of a bivalent equine papillomavirus type 2 (EcPV2)/BPV1 L1 VLP vaccine. Fourteen healthy horses were immunized with BPV1 L1 VLPs (100 µg per injection) plus adjuvant on days 0 and 28, while seven remained unvaccinated. On day 42, all 21 horses were challenged intradermally at 10 sites of the neck with 107 BPV1 virions per injection. In analogy, 14 horses immunized twice with EcPV2 plus BPV1 L1 VLPs (50 µg each) and seven control animals were challenged with 107 BPV2 virions per injection. Immunization with BPV1 L1 VLPs alone induced a robust antibody response (day 42 median titre: 12 800), and BPV1-inoculated skin remained unchanged in 13/14 vaccinated horses. Immunization with the bivalent vaccine was safe, resulted in lower median day 42 antibody titres of 400 for BPV1 and 1600 for EcPV2 and conferred significant yet incomplete cross-protection from BPV2-induced tumour formation, with 11/14 horses developing small, short-lived papules. Control horses developed pseudo-sarcoids at all inoculation sites. The monovalent BPV1 L1 VLP vaccine proved highly effective in protecting horses from BPV1-induced pseudo-sarcoid formation. Incomplete protection from BPV2-induced tumour development conferred by the bivalent vaccine is due to the poorer immune response by immune interference or lower cross-neutralization titres to heterologous BPV2 virions.

Bovine papillomavirus type 13 (BPV-13), a novel Deltapapillomavirus, has been found associated with urothelial tumours of the urinary bladder of cattle grazing on lands infested with bracken fern. BPV-13 was detected in 28 of 39 urothelial tumours. Diagnosis was based on sequencing of L1 and E5 amplicons from tumour samples. The nucleotide sequences generated from these amplicons showed a 100% homology with the sequences of BPV-13 L1 and E5 DNA found in Brazil from a fibropapilloma of the ear in a cow and from equine sarcoids in two horses. GenBank accession number of our representative BPV-13 sequences is JQ798171.1. Furthermore, mRNA encoding BPV-13 E5 oncoprotein was also documented, and its expression was also shown by immunohistochemistry and immunofluorescence in the basal and suprabasal urothelial tumour cells. In twenty-three tumours, BPV-13 was simultaneously found with BPV-2, a Deltapapillomavirus genus, species 4. The latter virus was detected by amplifying and sequencing a 154-bp-sized DNA fragment of BPV-2 E5. In addition, BPV-13 by itself was seen to be expressed in five BPV-2-negative urothelial tumours. This study shows that BPV-13 is present in urothelial tumour cells thus sharing biological properties with BPV-1 and BPV-2. Although further studies are needed, BPV-13 appears to be another worldwide infectious agent responsible for a distressing disease causing severe economic losses in cattle industry., (© 2015 Blackwell Verlag GmbH.)

Equine sarcoids develop upon bovine papillomavirus type 1 or 2 (BPV1, BPV2) infection in conjunction with trauma and represent the most common tumour disease in horses and other equids, including donkeys. In face of a sarcoid outbreak involving 12 of 111 donkeys and mules at the 'Rifugio degli Asinelli', a subsidiary charity organization of The Donkey Sanctuary, non-invasively collected sample material including crusts, dandruff, swabs and hair roots was collected from sarcoid-affected and 26 healthy donkeys, as well as dandruff from a grooming kit and tabanids caught from or in the vicinity of sarcoid patients. In addition five previously collected sarcoids stored in formalin were provided. DNA isolated from collected material was tested for the presence of the BPV1/2 E5 oncogene using PCR. Positive samples were further analysed by E2/E4 and LCR PCR and amplicon sequencing to determine a possible common source of infection via comparative alignment of intralesional BPV1/2 gene variants. IC/PCR was used to assess sample aliquots for the presence of BPV1/2 virions, and IHC to analyse five tumours for BPV1 E5 and L1 protein expression. All sarcoid-affected donkeys, two of 55 tabanids and dandruff from a curry comb tested positive for BPV1/2 E5, yet negative by IC/PCR. Healthy animals were BPV1/2-free. IHC revealed different levels of intralesional E5 and L1 expression. A series of BPV1 E5, E2, and LCR variants and BPV2 E5 were detected from donkeys, indicating that they had accidently developed sarcoids at about the same time rather than having acquired disease from each other., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Papillomas occur more frequently in cattle than other domestic animals. The causal agent of bovine papillomatosis is a virus that belongs to the family Papillomaviridae. In Tamaulipas, Mexico, the virus is considered a serious problem and has impeded the export of cattle to the United States, resulting in serious economic losses. Owing to the lack of information regarding the subtypes of papillomaviruses that infect cattle in Mexico, the aim of this study was to determine the subtypes in Tamaulipas. Fifty-two warts were analyzed with the use of polymerase chain reaction (PCR) involving primers that amplify the E7 gene of bovine papillomavirus (BPV). The PCR products were sequenced to differentiate the BPV-1 and BPV-2 subtypes. The sequencing quality was determined with the use of MEGA 6.0 software. Comparison of the Tamaulipas sequences with those of known BPV types by means of the MUSCLE algorithm showed that 53% of the former were BPV-1 and 47% were BPV-2. The distribution of the 2 subtypes in the cattle was homogeneous. This study demonstrated the presence of BPV-1 and BPV-2 in cattle from Tamaulipas and constitutes the first molecular characterization of papillomas in Mexico.

Although papillomaviruses (PVs) have been widely reported in vertebrates, there have been only a few PV reports in yaks (Bos grunniens). In 2012, Bam et al. reported bovine papillomavirus type 1 (BPV-1) and BPV-2 associated with cutaneous papillomatosis in yaks, which provided genomic and pathology information for yak PVs. However, nucleotide identity and phylogenic analyses revealed that there are two isolates with a high possibility of belonging to a novel type that is not BPV-1. The argument was thought to be caused by type-specific primers. Our analysis showed that BPV-1 type-specific primers can detect not only BPV-1 but also other PVs. It suggests that identification results using type-specific primers should be confirmed with more robust methods in molecular epidemiological studies.

Bovine papillomaviruses (BPV1/BPV2) have long been associated with equine sarcoids; deciphering their contribution has been difficult due to their ubiquitous presence on skin and in the environment, as well as the lack of decent techniques to interrogate their role in pathogenesis. We have developed and characterized an in situ hybridization (ISH) assay that uses a pool of probes complementary to portions of the E5, E6, and E7 genes. This assay is highly sensitive for direct visualization of viral transcript and nucleic acid in routinely processed histopathologic samples. We demonstrate here the visualization of BPV nucleic acid in 18 of 18 equine sarcoids, whereas no detectable viral DNA was present in 15 of 15 nonsarcoid controls by this technique. In nearly 90% (16/18) of the sarcoids, 50% or more of the fibroblastic cell nuclei distributed throughout the neoplasm had detectable hybridization. In the remaining 2 cases, fewer than half of the fibroblastic cells contained detectable hybridization, but viral nucleic acid was also detected in epithelial cells of the sebaceous glands, hair follicles and epidermis. A sensitive ISH assay is an indispensable addition to the molecular methods used to detect viral nucleic acid in tissue. We have used this technique to determine the specific cellular localization and distribution of BPV in a subset of equine sarcoids., (© The Author(s) 2015.)

The complete genome sequence of bovine papillomavirus 2 (BPV2) from Brazilian Amazon Region was determined using multiple-primed rolling circle amplification followed by Illumina sequencing. The genome is 7,947 bp long, with 45.9% GC content. It encodes seven early (E1, E2,E4, E5, E6,E7, and E8) and two late (L1 and L2) genes. The complete genome of a BPV2 can help in future studies since this BPV type is highly reported worldwide although the lack of complete genome sequences available.

Bovine papillomavirus (BPV) is a diverse group of double-stranded DNA oncogenic viruses. BPVs are classically described as epitheliotropic, however, they have been detected in body fluids, such as blood and semen. The presence of BPV in these sites can have implications for the dissemination of BPV. The aim of this study was to verify the prevalence of BPV types in cattle blood. A total of 57 blood samples were analyzed by PCR using BPV type-specific primers to BPVs 1-6 and 8-10, and subsequent sequencing. Sequencing quality was determined using Staden package with Phred 20. Similarity analysis was performed with BioEdit and BLAST programs to assess the identity with known BPV types. Statistical analysis was performed by Fisher's exact test. The results showed seven different types of BPVs in the blood, with the exception of BPV 5 and 9. This is the first study that demonstrates BPVs 3, 6, 8 and 10 DNA in cattle blood. BPVs 1 and 2 were the viral types most frequent in blood, while BPVs 4 and 10 were the least frequent types. All the samples showed co-infection by at least two BPV types. These data suggest that several BPV types may infect blood cells at the same time and demonstrate the possibility that the BPV infection in non-epithelial tissue can occur without restriction to one or two viral types. These results can contribute to future studies aimed at the control and prevention of papillomaviruses., (© 2014 Blackwell Verlag GmbH.)

Bovine papillomavirus (BPV) is an oncogenic virus with mucous and epithelial tropism. Possible productive virus infection in other tissues, such as blood, has been hypothesized. In order to investigate this possibility, three samples of skin papillomas and blood were collected from bovines with BPV infection and five samples of peripheral blood and one sample of normal tissue were collected from a calf without BPV infection. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood and examined by reverse transcription-polymerase chain reaction, immunofluorescence, in situ hybridization, and electron microscopy. The tissue samples were examined for histopathological and immunohistochemical features. The skin papillomas showed the presence of DNA sequences of BPV-2, BPV-11, and a putative virus type. The blood samples showed DNA sequences of BPV-1, 2, and 4 simultaneously. Immunohistochemistry showed BPV L1 protein in both epithelium and stroma and BPV E2 protein in koilocytes. In situ hybridization confirmed the presence of BPV DNA in PBMCs and immunofluorescence showed nuclear labeling of E2 and L1 BPV proteins in PBMCs. The transcription analysis revealed transcripts of BPV-1 L1, BPV-2 L2, and BPV-4 E7 in blood and papilloma samples of BPV-infected cattle. The comet assay revealed high levels of host cell DNA damage upon BPV infection. Electron microscopy analysis of PBMCs identified the presence of particles in the cytoplasm that are consistent with papillomavirus in size and shape. The productive infection of PBMCs with BPV has been previously discussed and this study provides evidence indicating that PBMCs are a target of BPV.

BPV-1 is known as the main causative agent of equine sarcoid, but the virus has also been detected in skin and blood of healthy horses. Previous reports demonstrated the presence of E5 variants in sarcoids of donkeys and horses; we investigated whether this genetic variability might be also found in BPV-1, PBMC associated, of sub-clinically infected horses. With this aim, we analyzed the E5 gene of 21 BPV-1 strains from diseased and sub-clinically infected horses. Our analyses lead us to demonstrate that multiple sequence variants can be present in the blood of sub-clinically infected horses, with alternative bases corresponding to either synonymous or non-synonymous codons in the E5 oncogene sequences. The results give support to the proposed existence of "equine adapted" BPV-1 strains with the occurrence of viral variants, resembling quasispecies, in clinically healthy horses with viremia., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Papillomaviruses are a family of slowly evolving DNA viruses and their evolution is commonly linked to that of their host species. However, whilst bovine papillomavirus-1 (BPV-1) primarily causes warts in its natural host, the cow, it can also cause locally aggressive and invasive skin tumours in equids, known as sarcoids, and thus provides a rare contemporary example of cross-species transmission of a papillomavirus. Here, we describe the first phylogenetic analysis of BPV-1 in equine sarcoids to our knowledge, allowing us to explore the evolutionary history of BPV-1 and investigate its cross-species association with equids. A phylogenetic analysis of the BPV-1 transcriptional promoter region (the long control region or LCR) was conducted on 15 bovine and 116 equine samples from four continents. Incorporating previous estimates for evolutionary rates in papillomavirus implied that the genetic diversity in the LCR variants was ancient and predated domestication of both equids and cattle. The phylogeny demonstrated geographical segregation into an ancestral group (African, South American and Australian samples), and a more recently derived, largely European clade. Whilst our data are consistent with BPV-1 originating in cattle, we found evidence of multiple, probably relatively recent, cross-species transmission events into horses. We also demonstrated the high prevalence of one particular sequence variant (variant 20), and suggest this may indicate that this variant shows a fitness advantage in equids. Although strong host specificity remains the norm in papillomaviruses, our results demonstrate that exceptions to this rule exist and can become epidemiologically relevant., (© 2014 The Authors.)

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., (© 2012 Blackwell Verlag GmbH.)

The aim of the study was to evaluate a novel diagnostic scheme which combines quantitative PCR and High-Resolution Melting (qPCR-HRM) curve analysis for rapid differentiation based on E5 partial CDS of bovine papillomavirus type 1 or 2 (BPV-1 or BPV-2), and to perform a phylogenetic analysis of the complete CDS of the E5 gene of BPV detected in equine sarcoids. Samples of 38 skin lesions obtained from 27 horses were collected for molecular examinations. All lesions were clinically diagnosed as sarcoids, but results of histopathological examinations did not always corroborate the clinical diagnosis. Although all the samples were positive for the presence of BPV DNA, after qPCR-HRM analysis 6 (16%) specimens were recognized as BPV-1 "wild", 24 (63%) as BPV-1 "European" and 8 (21%) as a "variant" of BPV E5 ORF partial CDS. Phylogenetic analysis based on nucleotide sequences of E2 ORF partial CDS and E5 ORF complete CDS was conducted on 7 specimens, whose sequences were published in GenBank and recognized as: 2PL (Accession Number --Acc. No. KC684939)--"variant" BPV-1, 7aPL (Acc. No. KC684940)--"European" BPV-1, 10PL (Acc. No. KC693480)--"variant" BPV-1, 16PL (Acc. No. KC693484)--"variant" BPV-2, 17PL (Acc. No. KC693481)--"variant" BPV-1, 20aPL (Acc. No. KC693482)--"European" BPV-1 and 20cPL (Acc. No. KC693483)--"wild" BPV-1. Amino acid (aa) sequences of E5 ORF complete CDS were also analyzed. The E5 variant of aa sequences found in isolate 10PL (protein identification--ID: AGM 20700) is a novel variant of E5 ORF complete CDS of BPV-1 detected in equine sarcoid in Poland.

Sarcoids are the most common skin tumors seen in horses worldwide. The pathogenesis of sarcoids is multifactorial, including an association with bovine papillomavirus types 1 and 2 and a genetic susceptibility to tumor development. Clinical manifestations vary and include occult, verrucous, nodular, fibroblastic, mixed, and malignant (malevolent) types. The tumor is nonmetastasizing but can become very aggressive locally. Multiple tumors are common. All clinical types can be present in the same horse. No treatment protocol is universally effective. The tumor has a high risk of recurrence. Recurrent and large tumors are associated with poorer prognoses., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Seven clinical cases of cutaneous papillomatosis in yaks were studied in Arunachal Pradesh, India. Sporadic, single or a chain of multiple varying size warts appeared around the eyes or on the body. Predominant site of warts was around eyes. Histopathologically, these cases were diagnosed as fibropapilloma. It was confirmed by the detection of BPV-1 and BPV-2 or their mixed infection by PCR and sequencing. Quantitative SYBR Green real-time PCR detected comparatively lower viral DNA copy number in cutaneous warts (CWs). Cases of CWs and its causative agent as bovine papillomavirus (BPVs) are reported for the first time in yaks., (© 2012 Blackwell Verlag GmbH.)

Sarcoids are common skin tumours of horses and donkeys that are characterised by persistent proliferation of dermal fibroblasts associated with the presence of bovine papillomavirus (BPV) DNA. Some early BPV proteins have been demonstrated within sarcoids and RNA containing both early and late transcripts is present, yet it remains unclear whether late replication of BPV, culminating in the production of infectious virus particles, can occur in equids. Here we report that BPV1 RNA isolated from equine sarcoids encodes a unique deletion of four residues within the L2 protein suggesting a novel variant of virus has evolved in equines. Such viral evolution would require the production and transmission of virus particles among horses with sarcoids. Quantitative RT-PCR demonstrated the presence of mRNA transcripts containing early gene message in sarcoid tissues and BPV-E2 early virus antigen was detected by immunofluorescence in the nuclei of dermal fibroblasts, but no E2 expression could be detected within the overlying epidermis where productive virus replication would be expected to occur. Although immunohistochemistry clearly detected late virus proteins in the nuclei of dermal cells from samples of bovine papillomas, no late protein expression was detected in formalin-fixed tissue from equine sarcoids; either in the dermis or epidermis. Moreover, quantitative RT-PCR demonstrated that late gene mRNA represented <0.3% of the transcribed BPV RNA. We conclude that BPV does not undergo productive infection in the epidermis overlying equine sarcoids at levels comparable with that occurring in its natural bovine host., (Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.)

Papillomaviruses (PV) are double-stranded DNA viruses that can cause benignant and malignant tumors in amniotes. There are 13 types of bovine papillomavirus (BPV-1 to -13); they have been found in reproductive tissues and body fluids. Normally these viruses are detected in epithelial tissue. We looked for BPV in the blood of healthy cattle and cattle with papillomatosis, using PCR and RT-PCR. BPV types 1 and 2 were detected in 8/12 blood samples of asymptomatic bovines and in 8/9 samples from cattle with papillomatosis. Six of 8 asymptomatic samples positive for BPV also showed expression for BPV. Five of 6 samples were positive for E2 expression, while 3/6 samples were positive for E5 expression. Five of 8 symptomatic samples positive for BPV also showed BPV expression. Five of 5 were positive for E2 expression, while 1/5 was positive for E5 expression. Two of 6 blood samples of asymptomatic cattle and 1/5 symptomatic blood samples scored positive for both E2 and E5 expression. This is the first study showing expression of BPV genes in the blood of asymptomatic and papillomatosis-affected animals.

Bovine papillomavirus (BPV) is an oncogenic virus related to serious livestock diseases. Oncoproteins encoded by BPV are involved in several steps of cellular transformation and have been reported as presenting clastogenic effects in peripheral lymphocytes and primary culture cells. The aim of this study was to evaluate the clastogenic potential of BPV types 1, 2, and 4 by comet assay. Peripheral blood was collected from 37 bovines, 32 infected with different levels of papillomatosis (12 animals have no affection) and five calves, virus free (negative control). The viral identification showed presence of more than one virus type in 59.375% of the infected animals. Comet assay was performed according to alkaline technique. The Kruskal-Wallis test showed statistical difference between the negative control group and infected animals (P = 0.0015). The Dunn post hoc test showed difference comparing the infected animals with calves. Mann-Whitney U test verified no difference between animals infected with only one viral type and animals presenting more than one viral type. The comet assay is considered an efficient tool for assessment of damage in the host chromatin due to viral action, specifically highlighting viral activity in blood cells.

A new electrochemical DNA biosensor for bovine papillomavirus (BPV) detection that was based on screen-printed electrodes was comprehensively studied by electrochemical methods of cyclic voltammetry (CV) and differential pulse voltammetry (DPV). A BPV probe was immobilised on a working electrode (gold) modified with a polymeric film of poly-L-lysine (PLL) and chitosan. The experimental design was carried out to evaluate the influence of polymers, probe concentration (BPV probe) and immobilisation time on the electrochemical reduction of methylene blue (MB). The polymer poly-L-lysine (PLL), a probe concentration of 1 μM and an immobilisation time of 60 min showed the best result for the BPV probe immobilisation. With the hybridisation of a complementary target sequence (BPV target), the electrochemical signal decreased compared to a BPV probe immobilised on the modified PLL-gold electrode. Viral DNA that was extracted from cattle with papillomatosis also showed a decrease in the MB electrochemical reduction, which suggested that the decreased electrochemical signal corresponded to a bovine papillomavirus infection. The hybridisation specificity experiments further indicated that the biosensor could discriminate the complementary sequence from the non-complementary sequence. Thus, the results showed that the development of analytical devices, such as a biosensor, could assist in the rapid and efficient detection of bovine papillomavirus DNA and help in the prevention and treatment of papillomatosis in cattle., (Copyright © 2012 Elsevier B.V. All rights reserved.)

This study was conducted with the objectives of detecting bovine papillomavirus type 2 (BPV-2) in urine samples and urinary bladder lesions in bovines using polymerase chain reaction (PCR) and real-time PCR-based molecular diagnostic tests, and quantifying BPV-2 in urinary bladder lesions especially in enzootic bovine haematuria (EBH)-affected animals. BPV-2 viral DNA was detected in urine samples (50%) and urinary bladder tissue (68.6%). Cloning and sequencing results showed a close homology with other Indian BPV-2 sequences. Quantitative real-time PCR (SYBR Green assay) showed that the BPV-2 load was low and similar irrespective of inflammatory or neoplastic lesions in the bladder. It was concluded that BPV-2 DNA is frequently present in urine and urinary bladder lesions in cows in an EBH endemic region and virus load was low in urinary bladder lesions., (© 2011 Blackwell Verlag GmbH.)

Papillomaviruses are found in epithelial lesions and are linked to different carcinogenic processes in humans and other animals. Although bovine papillomavirus (BPV) has been characterized as epitheliotropic, the presence of viral DNA has been detected in other sample types, including fresh semen. The aim of this study was to evaluate the presence of BPV DNA in spermatozoa and seminal plasma samples of commercial frozen semen taken from bulls (Bos taurus) and its effects on semen function. PCR assays were conducted with specific primers to detect BPV types 1-6 in 40 semen samples of dairy Gir bulls. The semen quality was assessed by the use of parameters such as motility, vigor, acrosomal integrity and DNA integrity. BPV-2 DNA was detected in all of the sperm cell samples and all the seminal samples; however BPV-1, 3, 4, 5 and 6 could not be detected. The presence of BPV DNA was apparently not a cause of reduced sperm function. This is the first record of BPV-2 DNA the commercial frozen semen taken from dairy Gir cattle by several companies that provide semen. Further studies are needed to assess the viability of the virus and the extent to which it can be spread through semen., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Skin lesions associated with papillomaviruses have been reported in many animal species and man. Bovine papillomavirus (BVP) affects mainly the epidermis, but also the dermis in several species including bovine, the best-known example being equine sarcoid, which is associated with BVP types 1 and 2. This publication describes and illustrates the macroscopic and histological appearance of BPV-associated papillomatous, fibropapillomatous or sarcoid-like lesions in Cape mountain zebra (Equus zebra zebra) from the Gariep Dam Nature Reserve, 2 giraffes (Giraffa camelopardalis) from the Kruger National Park, and a sable antelope (Hippotragus niger) from the Kimberley area of South Africa. An African buffalo (Syncerus caffer) cow from Kruger National Park also had papillomatous lesions but molecular characterisation of lesional virus was not done. Immunohistochemical staining using polyclonal rabbit antiserum to chemically disrupted BPV-1, which cross-reacts with the L1 capsid of most known papillomaviruses, was positive in cells of the stratum granulosum of lesions in Giraffe 1, the sable and the buffalo and negative in those of the zebra and Giraffe 2. Fibropapillomatous and sarcoid-like lesions from an adult bovine were used as positive control for the immunohistochemistry and are described and the immunohistochemistry illustrated for comparison. Macroscopically, both adult female giraffe had severely thickened multifocal to coalescing nodular and occasionally ulcerated lesions of the head, neck and trunk with local poorly-circumscribed invasion into the subcutis. Necropsy performed on the 2nd giraffe revealed neither internal metastases nor serious underlying disease. Giraffe 1 had scattered, and Giraffe 2 numerous, large, anaplastic, at times indistinctly multinucleated dermal fibroblasts with bizarre nuclei within the sarcoid-like lesions, which were BPV-1 positive in Giraffe 1 and BPV-1 and -2 positive in Giraffe 2 by RT-PCR. The sable antelope presented with a solitary large lesion just proximal to the right hind hoof, which recurred after excision, and was BPV-1 positive by RT-PCR. Other wart-like growths were present elsewhere on the body. The Cape mountain zebra either succumbed from their massive lesions or were euthanased or removed from the herd because of them. The lesions were BPV-1 and/or -2 positive by RT-PCR. The buffalo lesions were wart-like papillomatous projections in the inguinal and udder region. Stratum granulosum cells that stained immunohistochemically positive in the various species appeared koilocyte-like, as described in human papillomaviral lesions.

Papillomavirus was detected electron microscopically in cutaneous fibropapillomas of a giraffe (Giraffa camelopardalis) and a sable antelope (Hippotragus niger). The virus particles measured 45 nm in diameter. Histopathologically, the lesions showed histopathological features similar to those of equine sarcoid as well as positive immunoperoxidase-staining of tissue sections for papillomavirus antigen. Polymerase chain reaction (PCR) detected bovine papillomavirus (BPV) DNA. Bovine papillomavirus-1 was characterised by real-time PCR in the sable and giraffe, and cloning and sequencing of the PCR product revealed a similarity to BPV-1. As in the 1st giraffe, the lesions from a 2nd giraffe revealed locally malignant pleomorphism, possibly indicating the lesional end-point of papilloma infection. Neither virus particles nor positively staining papillomavirus antigen could be demonstrated in the 2nd giraffe but papillomavirus DNA was detected by real-time PCR which corresponded with BPV-1 and BPV-2.

Ten equine skin tumors that had been classified as schwannomas on routine histological examination were analyzed by polymerase chain reaction for bovine papillomavirus DNA. All 10 were positive for bovine papillomavirus 1 or 2, and all 10 were immunohistochemically negative for S-100 protein and strongly positive for vimentin. Nine tumors were moderately positive for laminin and 8, for smooth muscle actin. Five tumors were variably and weakly positive for type IV collagen. The lack of S-100 protein expression made Schwann cells an unlikely cell of origin, as opposed to peripheral nerve sheath tumors, which typically express S-100 protein, at least in some neoplastic cells. The immunohistochemical reactivity is consistent with myofibroblastic origin of the neoplastic cells, although smooth muscle cell or pericyte origin cannot be ruled out. These tumors represent an atypical form of equine sarcoid. Polymerase chain reaction for bovine papillomavirus and S-100 immunohistochemistry are strongly recommended for all equine skin tumors with histological characteristics typical of schwannoma or peripheral nerve sheath tumor.

Bovine digital dermatitis (BDD) is a common infectious foot disease whose aetiology is not fully understood. Its origin is thought to be multifactorial, with treponemes being involved. Using PCR-based techniques, BDD samples from 45 affected cows and intact skin from 8 BDD-affected and 33 healthy cows were assessed for the presence of bovine papillomavirus and Treponema DNA. BPV DNA (mainly BPV-1/2) was detected in 22% of lesions and one skin sample from affected animals, and in 15% (BPV-1/-2) and 23% (BPV-3/4/6/9/10) of skin samples from healthy cows. Using quantitative PCR, Treponema DNA was demonstrated in 38/45 BDD lesions, with bacterial DNA loads ranging between 2 × 10(3) and 2.78 × 10(5) copies/40 ng of total DNA. Qualitative PCR confirmed this result and revealed Treponema DNA in 4 additional BDD samples, thus leading to an overall infection rate of 93.3%. Sequence analysis of amplified Treponema DNA revealed T. pedis sp. nov. in 51%, T. medium ssp. bovis in 37.7%, and T. phagedenis ssp. vaccae in 4.4% of lesions. T. brennaborense was not detected in any of the samples. Six BDD samples contained type IV oral Treponema strains, 6 other harboured so far unpublished Treponema sequences. To our knowledge, this is the first report providing information on BPV infection in BDD-affected cattle, and the Treponema DNA load and occurrence of type IV treponemes in BDD samples. Our findings further support an etiologic association of treponemes, particularly T. pedis sp. nov., with BDD disease, yet indicate that BPVs do not directly contribute to BDD development., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Reasons for Performing the Study: Equine hoof canker is a chronic proliferative pododermatitis of as yet unknown aetiology. Like equine sarcoid disease, canker is a therapy-resistant disorder characterised by hyperkeratosis, acanthosis and a marked tendency to recur., Hypothesis: There is an association of sarcoid-inducing bovine papillomaviruses of types 1 and 2 (BPV-1, BPV-2) with hoof canker disease., Methods: Using PCR-based techniques, we assessed canker tissue, intact skin and/or peripheral blood mononuclear cells (PBMCs) of 25 canker-affected horses for the presence of sarcoid-associated BPV-1 and -2., Results: Conventional PCR revealed BPV-1/-2 DNA in 24/24 canker, 12/13 skin and 10/11 PBMC DNA isolates. Using inverse PCR, full-length BPV episomes were detected in 1/5 canker specimens. Sequencing of viral early and late genes amplified from canker, intact skin and PBMC DNA of 2 cases revealed an overall identity of 98% to BPV-1. Viral DNA loads amounted to ≤16 copies per cell in canker tissue and intact skin, and to ≤0.35 copies per PBMC, as determined by quantitative PCR. Using RT-PCR, the viral major oncogene E5 was shown to be transcribed in 2/4 canker tissue specimens and 5/7 PBMC isolates. Immunocapture PCR from 7 canker and 6 skin extract supernatants revealed capsomere-associated viral DNA in one canker and one skin sample. Hoof tissue, skin and PBMCs collected from 13 individuals with no signs of canker or BPV-related malignancies scored negative throughout the experiments., Conclusion: These findings suggest that the observed presence of BPV-1/-2 in canker-affected horses is not coincidental but indicative of an active contribution to hoof canker disease., Potential Relevance: The use of antivirals and/or immune modulators may help improving canker therapy., (© 2010 EVJ Ltd.)

Sarcoids are the most common tumor of the equine skin but only 1 study describing the epidemiology of sarcoids in Canadian horses has been published. The records of 5 veterinary diagnostic laboratories in western Canada were searched to identify submissions of sarcoids from horses. The submission records and diagnostic reports of 802 separate submissions of equine sarcoids were reviewed for age, breed, and gender of the horse and the number, location, and clinical type of sarcoid. From these records, the 307 submissions to laboratories in Saskatchewan were compared to a reference group to test for breed and gender predisposition. Based on clinical history and lesion descriptions, 5 clinical types of sarcoids were identified. Horses of various ages and 23 equine breeds were affected; donkeys were over-represented. Polymerase chain reaction (PCR) for the bovine papillomavirus (BPV) DNA was performed on formalin-fixed paraffin-embedded tissues from a stratified subset of 96 of the different clinical types; BPV2 was present in 60 of 74 (81%) for which a PCR product was obtained. Unlike other areas in the world, in western Canada, equine sarcoids are most commonly associated with BPV type 2.

Bovine cutaneous warts (CWs) were investigated in Northern India. Of 49 cases, 44 were recorded in cattle and 5 in buffaloes. These animals had mild to moderate grade infections. Grossly, cases of CWs appeared to be of exophytic type, however, different types of growth patterns were observed. A total of 26 biopsies (cattle 21 and buffaloes 5) from CWs-affected animals studied histopathologically were diagnosed as exophytic and cauliflower-like fibropapilloma 13, exophytic and dome-shaped fibropapilloma 5, occult and/or fibroblastic type papilloma 3, cauliflower-like papilloma 3, endophytic fibropapilloma 1 and fibroma 1. On PCR analysis, 11 CWs and 2 normal skin samples showed BPV-1, -2 mixed infections. A rapid, sensitive and reliable real-time SYBR Green PCR test to detect BPV-1, BPV-2 and to quantify BPV-1 was developed. Results of amplification and dissociation plot of real-time PCR revealed that six samples were BPV-1 positive, eight were BPV-2 positive and six were positive for both BPV-1 and -2. CWs samples from different dairy farms testing positive for BPV-1 by PCR assay were also positive using Quantitative real-time SYBR Green PCR assay. For the first time, mixed infection of BPV-1 and -2 was detected in India and BPV-1 load was quantified by real-time SYBR Green PCR assay.

Reasons for Performing Study: Sarcoids are nonmetastasising, yet locally aggressive skin tumours that constitute the most frequent neoplasm in equids. Infection by bovine papillomaviruses types 1 and 2 (BPV-1, BPV-2) has been recognised as major causative factor in sarcoid pathogenesis, but a possible correlation of intralesional virus load with disease severity has not been established thus far., Hypothesis: Given the pathogenic role of BPV-1 and BPV-2 in sarcoid disease, we suggest that intralesional viral DNA concentration may reflect the degree of affection., Methods: Severity of disease was addressed by recording the tumour growth kinetics, lesion number and tumour type for 37 sarcoid-bearing horses and one donkey. Viral load was estimated via quantitative real-time PCR (qPCR) of the E2, E5, L1 and L2 genes from the BPV-1/-2 genome for one randomly selected lesion per horse and correlated with disease severity., Results: Quantitative PCR against E2 identified viral DNA concentrations ranging from 0-556 copies/tumour cell. Of 16 horses affected by quiescent, slowly growing single tumours or multiple mild-type lesions, 15 showed a viral load up to 1.4 copies per cell. In stark contrast, all equids (22/22) bearing rapidly growing and/or multiple aggressive sarcoids had a viral load between 3 and 569 copies per cell. Consistent results were obtained with qPCR against E5, L1 and L2., Conclusions: While tumours of the same clinical type carried variable virus load, confirming that viral titre does not determine clinical appearance, we identified a highly significant correlation between intralesional viral load and disease severity., Potential Relevance: The rapid determination of BPV viral load will give a reliable marker for disease severity and may also be considered when establishing a therapeutic strategy.

BPV-1 is now recognized as a main etiological agent of equine sarcoids. The etiopathogenesis of the equine sarcoids is equivocal and is not yet fully understood. The aim of the present study was to analyse a partial sequence of the L1 gene of BPV associated with equine sarcoids in Polish horses. After clinical diagnosis, 40 skin lesions obtained from 29 horses were collected. The amplicons of a fragment of BPV L1 DNA were detected using PCR with MY09/MY11 primers in 31 specimens. All of them were recognized as BPV-1. Phylogenetic analysis has allowed the amplicons of partial L1 gene to be divided into 3 phylogenetic groups (A, B, C) and one separate isolate (20c). Sequence variants from phylogenetic groups B, C and isolate 20c represented new genetic variants of BPV-1 L1. Sequence variants from groups B and C were submitted to GenBank NCBI.

Infection with bovine papillomavirus type 1 (BPV1) or BPV2 induces fibropapillomas in cows and skin sarcoids in horses. Prophylactic vaccination targeting BPV1 and BPV2 may reduce the incidence of these economically important diseases. The L1 major capsid proteins of BPV1 and BPV2 were expressed in Sf-9 insect cells and both self-assembled into virus-like particles (VLPs). Using conformation-dependent monoclonal antibodies (mAb) both type-specific and shared epitopes were detected. Antisera were raised against BPV1 or BPV2 VLP using alum adjuvant, and their (cross)neutralization capacity was tested by C127 neutralization assays using native BPV1 and BPV2 virions, or by BPV1 pseudovirion assay. Antisera induced by either VLP vaccine were able to robustly (cross-)neutralize heterologous as well as homologous types, indicating that BPV1 and BPV2 are closely related serotypes. These results suggest that a monovalent BPV1 (or BPV2) VLP vaccine may potentially protect against both BPV1 and BPV2 infections and associated diseases.