Performing Department
Veterinary Clinical Sciences
Non Technical Summary
Equine sarcoids are locally invasive skin tumors and represent the most common tumor in equids worldwide. Sarcoids are classified according to their appearance and behavior, with categories including occult (flat), verrucous (wart-like), nodular, fibroblastic (ulcerated), and malevolent (aggressive). Sarcoids can develop in any location, either as a single tumor or as multiple tumors of different type. Although sarcoid tumors do not spread to other organs, they can significantly impact the function and aesthetics of affected equids based on tumor location, size and frequency. Thus, the value of horses with sarcoids is often dramatically decreased. Sarcoid tumors may cause discomfort and can result in ulceration, infection, and occasionally lameness associated with lesion location. Bovine papillomavirus types 1 and 2 (BPV-1, BPV-2) are necessary for sarcoid development, but can also be found in normal horse skin. Quarter Horses, Arabians and Appaloosas have an increased risk for sarcoid development, suggesting a genetic predisposition. Therefore, genetic markers from Quarter Horses with sarcoid will be compared to Quarter Horses without sarcoid. We expect that horses with sarcoid will be genetically different from horses without sarcoid. If this is true, Quarter Horses with the "sarcoid-susceptible" genes will be used for further studies; if not, Quarter Horses will still be used because of their increased risk of sarcoid development compared to other breeds. The next objective is to develop a BPV virus inoculum that when injected into the skin of a genetically-susceptible horse, causes a sarcoid. This inoculum will be grown in the lab, and then injected in escalating doses into the skin of Quarter Horses without sarcoid but with the susceptible genes (if identified above). The dose of inoculum that causes the longest-lasting, largest sarcoids will be selected for vaccine studies. Next, virus-like particles (VLP) that are similar to natural BPV but are non-infectious due to the absence of viral DNA will be used as a vaccine. This VLP-based vaccine will be tested for prophylactic efficacy in BPV-susceptible horses, following a vaccine safety and immune response trial. Vaccine efficacy will be determined by visual and histopathological examination of cutaneous lesions, as well as lymphocyte response tests. The final objective is to explore preventative strategies for recurrence following surgical removal of sarcoids. We suspect that the presence of BPV DNA in histologically tumor-free surgical margins is associated with risk of recurrence, and that increasing quantity of BPV DNA is associated with an increased risk of recurrence. Therefore, quantitation of BPV DNA in excised tumor margins will be used to determine the BPV type and the predictive value of PCR testing for recurrence. Taken together, this information highlights the need for clarification of how and why sarcoids develop to clearly identify at-risk populations. In addition, development of vaccines that can prevent sarcoids will decrease the incidence and severity of disease, and have important implications for papillomavirus infection in other species.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
The long-term goal of the proposed study is to elucidate the pathogenesis of equine sarcoid tumors and to implement effective preventative strategies for bovine papillomavirus (BPV), the etiologic agent of equine sarcoids. This cutaneous neoplasm represents the most common equine tumor, and is associated with the only documented case of natural cross-species papillomavirus infection. Tumors are characterized by locally invasive, transformed fibroblasts that are non-permissive for BPV production. This research proposal has four objectives, the first of which is to determine the basis for an apparent breed predilection. A retrospective analysis of sarcoid-bearing equids will be done to provide descriptive data and is expected to corroborate previous findings that Quarter Horses are most commonly affected. Genetic markers from sarcoid-bearing equids will be compared to nonsarcoid-bearing equids of the same breed. The second objective is to develop a BPV inoculum that will induce sarcoid lesions in susceptible horses. Adaptive immune responses to this experimental challenge virus will be evaluated. It is expected that an inoculum dose will be identified that will induce sarcoid-like lesions as well as humoral immune responses. This virus will then be used to test the efficacy of future prophylactic vaccine candidates. The third objective is to develop virus-like particles (VLP) that are morphologically and antigenically similar to natural BPV, but are non-infectious due to the absence of viral DNA. This VLP-based vaccine will be tested for immunoprophylactic efficacy in BPV-susceptible horses, following a vaccine safety and immunogenicity trial. Vaccine efficacy will be determined by visual and histopathological examination of cutaneous lesions, as well as lymphocyte response tests. We expect the vaccine to protect against sarcoid development in naive, susceptible horses following viral challenge. The final objective is to explore preventative strategies for recurrence following surgical excision of sarcoid tumors. Quantitation of BPV DNA in excised tumor margins will be used to determine the BPV type and the predictive value of PCR for recurrence. It is expected that increased BPV DNA in surgical margins will be positively associated with tumor recurrence.
Project Methods
An extensive retrospective analysis of equids affected by sarcoid will be performed to determine risk factors associated with disease by use of the Veterinary Medical Database. Next, we will determine the equine leukocyte antigen (ELA) haplotype distribution for major histocompatibility complex (MHC) Class I and Class II genes among sarcoid-bearing Quarter Horses and nonsarcoid-bearing Quarter Horses. Serum will be collected from 40 Quarter Horses without sarcoid and 40 age-matched Quarter Horses with sarcoid, selected from the PUVTH record database. The ELA haplotype for both MHC Class I and II genes will be determined by using polymorphic microsatellites and sequencing. Cattle papillomas that contain BPV-1, as determined by PCR and sequencing, will be used to obtain infectious virions. Escalating doses of inoculum will be injected intradermally into nonsarcoid-bearing Quarter Horses with the predominant ELA haplotype identified in sarcoid-bearing horses. In the event that association between haplotype and sarcoid status is not identified, nonsarcoid-bearing Quarter Horses will be used regardless of ELA haplotype. Sarcoid-like lesions will be measured, visually categorized as occult, verrucous, nodular, fibroblastic or malevolent, and then biopsied for histopathological assessment and BPV-1 DNA quantification by real-time PCR. The humoral immune response to these injections will be measured. The inoculum dose that results in visible lesions in the majority of horses and persists for the longest period of time will be selected for future prophylactic vaccine studies. Virus-like particles which resemble natural virions will be generated for use as vaccines. Nonsarcoid-bearing Quarter Horses will be inoculated with escalating doses of VLP, followed by lymphocyte response tests. The VLP dose that results in the most robust humoral immune response will be used for future prophylactic vaccine studies. For assessment of prophylactic use of VLP, a randomized, blinded, controlled study will be performed. Horses with a BPV-susceptible ELA haplotype or Quarter Horses will be used in this trial. Horses will receive the VLP vaccine at a dose determined by neutralizing antibody induction for three injections, followed by intradermal infectious virion challenge. Vaccine efficacy will be determined by visual and histopathological examination of lesions, quantitative viral DNA analysis at virion inoculation sites, and serum neutralizing antibody responses. If humoral immune responses to VLP are weak, the AS04 adjuvant will be tested for its ability to enhance these responses. Finally, to test whether presence of BPV DNA in histologically tumor-free surgical margins of sarcoid tumors is associated with recurrence, margins of sarcoid tumors deemed completely resected by histopathology will be tested for BPV DNA by real-time PCR.