Lies Bogaert

Distal limb cast sores in horses: risk factors and early detection using thermography.

REASONS FOR PERFORMING STUDY There is a lack of evidence-based data on the prevalence, outcome and risk factors of distal limb cast sores, and no objective tool has been described for the early detection of cast sores. OBJECTIVES To investigate the prevalence, location, outcome and risk factors of cast sores after application of a distal limb cast and to determine whether static thermography of the cast is a valuable tool for the assessment of sores. METHODS A prospective study was conducted on horses treated with a distal limb cast. At each cast removal, cast sores were graded as superficial sores (SS), deep dermal sores (DS) or full thickness skin ulcerations (FS). In several cases, a thermographic evaluation of the cast was performed immediately prior to removal and differences in temperature (AT) between the coolest point of the cast and 2 cast regions predisposed for sore development (dorsoproximal mc/mtIII and palmar/plantar fetlock) were calculated. RESULTS Mean +/- s.d. total casting time of 70 horses was 31 +/- 18 days. Overall, 57 legs (81%) developed at least SS. Twenty-four legs (34%) ultimately developed DS and one horse had an FS. Multivariable analysis showed that the severity of sores was positively associated with increasing age (OR: 1.111, P = 0.028), a normal (vs. swollen) limb (OR: 3387, P = 0.023) and an increase in total casting time (OR per week: 1.363, P = 0.002). The thermographic evaluation (35 casts) revealed that the severity of sores was positively associated with increasing deltaT (OR: 2.100, P = 0.0005). The optimal cut-off values for the presence of SS and DS were set at, respectively, deltaT = 23 and 43 degrees C. CONCLUSION AND POTENTIAL RELEVANCE Distal limb cast is a safe coaptation technique with increasing risk of developing sores with time. Thermography is a valuable and rapid clinical tool to monitor the development of cast sores.

Bovine papillomavirus DNA can be detected in keratinocytes of equine sarcoid tumors

Bovine papillomavirus (BPV)-1 and -2 is linked to equine sarcoids, a commonly observed skin tumor in horses that is of considerable veterinary importance. Previous studies using in situ hybridization have detected BPV DNA only in fibroblasts and not in keratinocytes of sarcoids. In contrast, normal equine skin latently infected with BPV shows a dysplastic epithelium without dermal changes, similar to lesions induced by other papillomavirus types infecting the epithelium. The first goal of our study was to describe the epidermal and dermal characteristics of several stages in sarcoid development. Next, we explored whether BPV can infect epidermal cells in the horse using real-time PCR on laser-micro-dissected keratinocytes and fibroblasts. We found that latently infected normal skin samples and a subset of early stage sarcoids show dysplastic, koilocyte-like epithelial changes. BPV DNA was detected in keratinocytes in 40% of the samples with these particular epithelial properties, whereas advanced sarcoids only had BPV DNA in the fibroblasts. These data may indicate a novel and intriguing pathway of BPV infection in the horse composed of a first step of keratinocyte infection, followed by migration of viral material towards the dermis resulting in infection of sub-epidermal fibroblasts and their fully transformed phenotype. Additionally, an example of co-existence of a dermal BPV-1 and an epidermal BPV-2 infection in the same lesion is shown, indicating that horses can harbor infection with more than one BPV type at the same time.

EcPV2 DNA in equine genital squamous cell carcinomas and normal genital mucosa

Squamous cell carcinoma (SCC) represents the most common genital malignant tumor in horses. Similar to humans, papillomaviruses (PVs) have been proposed as etiological agents and recently Equine papillomavirus type 2 (EcPV2) has been identified in a subset of genital SCCs. The goals of this study were (1) to determine the prevalence of EcPV2 DNA in tissue samples from equine genital SCCs, penile intraepithelial neoplasia (PIN) and penile papillomas, using EcPV2-specific PCR, (2) to examine the prevalence of latent EcPV2 infection in healthy genital mucosa and (3) to determine genetic variability within EcPV2 and to disentangle phylogenetic relationships of EcPV2 among PVs. EcPV2 DNA was detected in all but one penile SCC (15/16), in all PIN lesions (8/8) and penile papillomas (4/4). Additionally, EcPV2 DNA was demonstrated in one of two metastasized lymph nodes, one contact metastasis in the mouth, two vaginal and one anal lesion. In healthy horses, EcPV2 DNA was detected in 10% (4/39) of penile swabs but in none of vulvovaginal swabs (0/20). This study confirms the presence of EcPV2 DNA in equine genital SCCs and shows its involvement in anal lesions, a lymph node and contact metastases. Latent EcPV2 presence was also shown in normal male genital mucosa. We found that different EcPV2 variants cocirculate among horses and that EcPV2 is related to the Delta+Zeta PVs and is only a very distant relative of high-risk human PVs causing genital cancer. Thus, similar viral tropism and similar malignant outcome of the infection do not imply close evolutionary relationship.

Expression of LIGHT/TNFSF14 Combined with Vaccination against Human Papillomavirus Type 16 E7 Induces Significant Tumor Regression

LIGHT, a ligand for the lymphotoxin-beta receptor, establishes lymphoid-like tissues inside tumor sites and recruits naïve T cells into the tumor. However, whether these infiltrating T cells are specific for tumor antigens is not known. We hypothesized that therapy with LIGHT can expand functional tumor-specific CD8(+) T cells that can be boosted using HPV16E6E7-Venezuelan equine encephalitis virus replicon particles (HPV16-VRP) and that this combined therapy can eradicate human papillomavirus 16 (HPV16)-induced tumors. Our data show that forced expression of LIGHT in tumors results in an increase in expression of IFNgamma and chemoattractant cytokines such as interleukin-1a, MIG, and macrophage inflammatory protein-2 within the tumor and that this tumor microenvironment correlates with an increase in frequency of tumor-infiltrating CD8(+) T cells. Forced expression of LIGHT also results in the expansion of functional T cells that recognize multiple tumor antigens, including HPV16 E7, and these T cells prevent the outgrowth of tumors on secondary challenge. Subsequent boosting of E7-specific T cells by vaccination with HPV16-VRP significantly increases their frequency in both the periphery and the tumor and leads to the eradication of large well-established tumors, for which either treatment alone is not successful. These data establish the safety of Ad-LIGHT as a therapeutic intervention in preclinical studies and suggest that patients with HPV16(+) tumors may benefit from combined immunotherapy with LIGHT and antigen-specific vaccination.

Molecular and Immunohistochemical Distinction of Equine Sarcoid From Schwannoma

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.

EcPV2 DNA in equine squamous cell carcinomas and normal genital and ocular mucosa.

Squamous cell carcinoma (SCC) represents the most common malignant tumour of the eye and external genitals in horses. Comparable to humans, papillomaviruses (PV) have been proposed as etiological agents of cancer in horses and recently, Equine papillomavirus type 2 (EcPV2) has been identified in genital SCCs. Hitherto it had never been demonstrated in ocular SCCs. The first goal of this study was to determine the prevalence of EcPV2 DNA in tissue samples from equine genital and ocular SCCs, genital papillomas and penile intraepithelial neoplasia (PIN) lesions, using EcPV2-specific PCR. The second goal was to investigate the possibility of latent EcPV2 infection in the genital and ocular mucosa of healthy horses on swabs obtained from the eye, penis, vulvovaginal region and cervix. EcPV2 DNA was detected in all genital SCCs (17/17), genital papillomas (8/8), PIN lesions (11/11) and ocular SCCs (9/9). In healthy horses, EcPV2 DNA was detected in 43% (17/40) of penile swabs, 53% (9/17) of vulvovaginal swabs, 47% (8/17) of cervical swabs and 57% (32/56) of ocular swabs. This study confirms the presence of EcPV2 DNA in equine genital SCCs. Moreover, we demonstrate for the first time its involvement in other genital lesions and in ocular SCCs and latent EcPV2 infections in normal genital (including cervical) and ocular equine mucosa. The close relatives of EcPV2 are associated to cutaneous lesions, and this virus is not related to high-risk human papillomaviruses causing cervical cancer. Thus, similar viral tropism does not imply close evolutionary relationship.

Factors affecting outcome of extensor tendon lacerations in the distal limb of horses - A retrospective study of 156 cases (1994–2003)

Distal limb lacerations are common injuries in horses, with a better prognosis reported for extensor tendons lacerations compared to flexor tendons lacerations. The objective of the present study was to determine the influence of type and location of injury, modalities of treatment and post surgical complications on the outcome of extensor tendon lacerations. The medical records of 156 horses surgically treated for extensor tendon lacerations over a 10-year period were analysed retrospectively. Information was obtained for 124 horses with a minimum of 18 months follow-up. Statistical analysis was performed on 129 horses (five horses were euthanatized during hospitalization) in order to determine factors affecting outcome. Seventy-four percent of the horses returned to soundness, 17% had moderate gait impairment and 9% remained lame. Approximately 60% of the sports horses returned to an equal or higher level of performance, and 17.5% returned to a lower level. In the multivariate statistical analysis, the only significant factor that favourably influenced outcome was complete suture of the wound. A significant association could not be detected between outcome and absence of a functional extensor tendon, The most important post-surgical complication was extensive scarring of the wound. The present results report outcome of extensor tendon lacerations in a large number of horses and outline the importance of primary wound healing in order to avoid major scarring with potential functional consequences.

Osteochondral Fragmentation in the Synovial Pad of the Fetlock in Warmblood Horses

OBJECTIVES To determine clinical and arthroscopic characteristics associated with fragments in the synovial pad of the fetlock and to characterize their morphology. STUDY DESIGN Retrospective study. ANIMALS Warmblood horses (n=104) with fragment(s) in the synovial pad. METHODS Signalment and results of radiographic and clinical examination were collected before surgery. After arthroscopic fragment removal and joint evaluation for synovial and/or cartilage abnormalities, fragments were measured, and evaluated by histopathology. RESULTS Synovial pad fragments (n=142) were removed from 127 fetlocks. Two older horses had lameness. During arthroscopy, abnormalities were observed in 40 joints (31.5%) and multivariate logistic regression analysis showed a significant correlation between the observed arthroscopic abnormalities and the presence of large fragments (P=.016). Fragments were osteochondral bodies completely surrounded by fibrous tissue. At the edges of the hyaline cartilage cap an underlying fibrous structure was obvious in the extracellular matrix giving it a reactive pattern. CONCLUSIONS Although the impact on lameness was minimal, there was a significant correlation between arthroscopic abnormalities and presence of large synovial pad fragments. On histopathology, these osteochondral fragments are embedded in fibrous tissue and show a reactive pattern. They are not a manifestation of any well-described joint pathology. CLINICAL RELEVANCE Large synovial pad fragments in Warmblood horses can be associated with synovial and cartilage abnormalities, but further studies are warranted to determine their origin and clinical importance.

A novel murine model for evaluating bovine papillomavirus prophylactics/therapeutics for equine sarcoid-like tumours

Equine sarcoids are highly recurrent bovine papillomavirus (BPV)-induced fibroblastic neoplasms that are the most common skin tumours in horses. In order to facilitate the study of potential equine sarcoid prophylactics or therapeutics, which can be a slow and costly process in equines, a murine model for BPV-1 protein-expressing equine sarcoid-like tumours was developed in mice through stable transfection of BPV-1 E5 and E6 in a murine fibroblast tumour cell line (K-BALB). Like equine sarcoids, these murine tumour cells (BPV-KB) were of fibroblast origin, were tumorigenic and expressed BPV-1 proteins. As an initial investigation of the preclinical potential of this tumour model for equine sarcoids prophylactics, mice were immunized with BPV-1 E5E6 Venezuelan equine encephalitis virus replicon particles, prior to BPV-KB challenge, which resulted in an increased tumour-free period compared with controls, indicating that the BPV-KB murine model may be a valuable preclinical alternative to equine clinical trials.