Both PCR and IHC gave positive results for more than 50% of the equine papillomas studied. The proportion of positive samples was not significantly different with either test, although it must be remembered that IHC identifies only generic PV antigen, whereas PCR confirms the presence of EPV. Cohen’s kappa statistic indicated moderate agreement between the results of the 2 tests. All the negative- control samples were negative by both tests.
Amplification and identification of EPV DNA in equine cutaneous papillomas suggest that EPV is involved in the development of these lesions. Nucleotide substitutions may reflect polymerase-copying errors or inherited substitutions, indicating descent of these organisms from a common ancestor. The high degree of identity in the 384-bp nucleotide sequence suggests that the product amplified is EPV DNA, although our conclusions are based on a small segment of a 7610-bp genome. The amount of natural variation in genetic sequence between isolates of EPV is unknown.
Although the technique of DNA extraction from FFPE tissues is considered robust and most DNA can be extracted and amplified, preparations from fixed tissues always exhibit certain limitations for PCR. Formalin fixation can induce degradation of DNA, resulting in a reduction in test sensitivity. The PCR primers were designed to amplify a relatively small (384-bp) region to minimize the loss of sensitivity due to sheared DNA (26
). Fixation time has been shown to reduce amplification efficiency, especially when tissue is fixed in buffered 10% formalin for 1 to 4 wk; there is little or no effect when tissue is fixed for 48 h or less (27
). Although tissues are usually fixed for 48 h or less, the precise fixation time was unknown for the tissues in our study.
The presence of paraffin-embedded material or other contaminants in extracted DNA can inhibit DNA amplification, causing false-negative results (28
). Detection of amplifiable p53 DNA in our study confirmed that the DNA extraction protocol was adequate and ruled out the possibility of interference in the PCR assay, particularly inhibition of the Taq
polymerase, by contaminants in the DNA samples (30
). Age of the sample is also an important consideration: successful amplification has been reported in 40-y-old FFPE tissues, but researchers have reported a decline in amplification from fixed tissues that are 5 y old or older (30
). In our study, tissue blocks were up to 17 y old, although amplification of p53 DNA was demonstrated in tissue of various ages.
Depending on the developmental stage of the papilloma, EPV DNA may have been present at levels less than detectable by PCR. Studies of bovine and equine papillomas have shown that in the early infection and growth phase the lower layers of the squamous epithelium contain little PV DNA (1
), viral-protein expression and virus assembly being in the upper epidermal layers (1
). Another possible explanation for false-negative results is deletion or mutation within the region of EPV targeted by the PCR, resulting in failure of the primers to recognize the gene sequence (28
). It is possible that nested PCR would have increased the ability to amplify very low amounts of viral genome, thus reducing the risk of false-negative results. However, a greatly increased risk of contamination is associated with nested PCR, limiting its usefulness as a diagnostic tool.
Congenital papillomas have occasionally been reported in newborn foals (31
). Our study included 2 congenital papillomas; the absence of amplifiable EPV DNA and PV antigen is consistent with previous reports that showed no evidence of inclusion bodies and negative IHC results for PV (10
When the PCR products from the genital (penile and vulvar) papillomas in our study were sequenced, matches with EPV DNA nucleotide sequences were not found. These results are consistent with a previous report demonstrating that PV from 2 penile papillomas failed to hybridize with an EPV probe, suggesting the existence of a novel equine PV type (3
The primary antibody used in this study for IHC testing reacts with PV-specific common structural capsid antigens that are well conserved across species (2
). Since PV virions are assembled in the superficial keratinocytes (3
), IHC staining is visualized in the superficial layers of the squamous epithelium when replicating PV is present. Lack of detection of viral antigen in some of our equine papillomas may indicate that there was no replicating PV or that antigenicity was inhibited by fixation (23
). Nonproductive PV lesions, such as equine sarcoids, are not expected to contain viral antigen (2
); therefore, negative IHC results for the equine sarcoids in our study were expected. Since papillomas initially have basal cell hyperplasia without virion assembly, some papillomas may contain PV DNA but not antigen (10
), as found in 3 of the papillomas in our study.
Both the EPV-PCR and the consensus primers failed to amplify DNA in aural plaques, but PV antigen was detected in some, suggesting the possibility of a novel PV that differs sufficiently in nucleotide sequence to prevent the primers from binding. This finding is in agreement with a previous report (5
) that introduced the possibility of different types of EPV as etiologic agents for cutaneous papillomas and aural plaques. Differences between clinical manifestations of warts and aural plaques may be attributed to genetic variance between strains of EPV: it has been recognized that, within a given host species, different PV strains appear to preferentially affect different epithelial locations (35
The consensus primers allow the detection in FFPE tissues of DNA from PVs related to BPV1, BPV2, BPV5, ovine PV, European elk PV, deer PV, BPV3, BPV4, BPV6, canine oral PV, cottontail rabbit PV, and Human papillomavirus
(HPV) types 1, 41, and 63 (22
). Consensus primers amplified DNA in 2 of the papilloma tissues in our study. In the absence of successful sequencing, the DNA amplification was most likely nonspecific. Amplification with the consensus primers was also demonstrated in most of the equine sarcoids, and this was expected on the basis of reports that types 1 and 2 BPV are present in most sarcoids (16
). Given the large amount of connective tissue in certain types of sarcoids, it is possible that a particular section may contain little or no viral DNA and yield a false-negative result (36
). The presence of a PCR-positive result with the use of EPV primers in 1 sarcoid is difficult to interpret, as the sequencing results indicated homology within only a 150-bp region of the 384-bp sequence. The presence of a latent virus is a possibility, since the PV genome can often be found in normal epithelium, and normal epithelium is the accepted site of latent infection (34
). Although the sample size for sarcoids was small, if EPV is etiologically involved, we might have expected more positive results with the EPV primers.
In conclusion, we demonstrated EPV DNA in a high proportion of equine papillomas but not in aural plaques or sarcoids, which suggests that EPV may have a direct involvement in the pathogenesis of cutaneous papillomas. Nucleotide differences in EPV strains could explain the inability to detect EPV DNA in aural plaques; continued research is required to determine the cause of these lesions. Since PV demonstrates a higher degree of conservation within the E2
) than in the E4
), additional primers designed to amplify these regions may allow amplification of EPV DNA from a greater proportion of equine papillomas and aural plaques. In conjunction with IHC testing, PCR may be useful in confirming the presence of EPV in some equine papillomas, especially those in which histologic differentiation from other lesions is problematic.