Inoculation of young horses with bovine papillomavirus type 1 virions leads to early infection of PBMCs prior to pseudo-sarcoid formation.

Bovine papillomavirus types 1 and 2 (BPV-1 and BPV-2) are known to induce common equine skin tumours, termed sarcoids. Recently, it was demonstrated that vaccination with BPV-1 virus-like particles (VLPs) is safe and highly immunogenic in horses. To establish a BPV-1 challenge model for evaluation of the protective potential of BPV-1 VLPs, four foals were injected intradermally with infectious BPV-1 virions and with viral genome-based and control inocula, and monitored daily for tumour development. Blood was taken before inoculation and at weekly intervals. BPV-1-specific serum antibodies were detected by a pseudo-virion neutralization assay. Total nucleic acids extracted from tumours, intact skin and PBMCs were tested for the presence of BPV-1 DNA and mRNA using PCR and RT-PCR, respectively. Intralesional E5 oncoprotein expression was determined by immunofluorescence. Pseudo-sarcoids developed exclusively at sites inoculated with virions. Tumours became palpable 11-32 days after virion challenge, reached a size of ≤20 mm in diameter and then resolved in ≤6 months. No neutralizing anti-BPV-1 serum antibodies were detectable pre- or post-challenge. BPV-1 DNA was present in lesions but not in intact skin. In PBMCs, viral DNA was already detectable before lesions were first palpable, in concentrations correlating directly with tumour growth kinetics. PBMCs from two of two foals also harboured E5 mRNA. Immunofluorescence revealed the presence of the E5 protein in tumour fibroblasts, but not in the apparently normal epidermis overlying the lesions. Together with previous findings obtained in horses and cows, these data suggest that papillomavirus infection may include a viraemic phase.

BPV-1 infection is not confined to the dermis but also involves the epidermis of equine sarcoids.

In equids, bovine papillomaviruses of type 1 (BPV-1) and less frequently type 2 induce common, locally aggressive skin tumours termed sarcoids. Whereas BPV infection in cattle usually involves the epidermis and is productive in this skin layer, infection in equids is currently thought to be abortive, with virus solely residing as multiple episomes in dermal fibroblasts. Based on recent observations that do not agree with this assumption, we hypothesised that BPV also infects equid epidermis and is active in this skin layer. To test this hypothesis, we conducted a proof-of-principle study on eight distinct sarcoids. Presence of viral DNA was addressed by qualitative and quantitative BPV-1 PCR from microdissected sarcoid epidermis, and by subsequent amplicon sequencing. Viral activity was assessed by screening sarcoid epidermis for BPV-1 protein expression using immunohistochemistry (IHC) or immunofluorescence (IF). Virus-free equine skin served as negative control throughout the assays. BPV-1 DNA was demonstrated in all sarcoid epidermis samples, with viral DNA loads ranging between 2 and 195 copies/cell. Identical BPV-1 E5 genes were identified in epidermis and dermis of each of two sarcoids, yet different E5 variants were found in individual lesions. IHC/IF revealed the presence of E5 and E7 protein in sarcoid epidermis, and L1 capsomers in the squamous layer of one lesion. These findings indicate that BPV infection also involves the epidermis, where it may occasionally be productive.

Prevalence of bovine papillomavirus and Treponema DNA in bovine digital dermatitis lesions.

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.