Detection of Bovine Papillomaviruses (BPVs) and Immunohistochemical Expression of Ki-67 in Cutaneous Warts of Cattle and Buffaloes

Bovine papillomatosis is readily observed condition in cattle and buffaloes. The location and severity of the infection plays a crucial role to access its impact on the productivity and economic loss. The aim of present research was to study the association of different BPV types alone or their mixed infections in cutaneous warts (CWs) in cattle and buffaloes and immunohistochemical expression of Ki-67 in the CWs. A total of 33 CWs cases (Cattle, 23 and Buffaloes, 10) were studied. Grossly, the CWs were of varying size and shape (rough, irregular cauliflower-like growths with many finger-like horny processes on the surface and elevated dome shaped growths) and were attached to the skin by a broad base. Histopathologically, these were diagnosed as papilloma, fibropapilloma, fibroma and fibrosarcoma. Fibropapilloma was the most frequent histological type diagnosed. Histopathological findings in papilloma and fibropapilloma were characterized by presence of hyperkeratosis, parakeratosis and varying degrees of epidermal hyperplasia with irregular papillary projections into the dermis. Moderate to severe acanthosis, basophilic keratohyaline granules and koilocytes with condensed hyperchromatic central, eccentric or crescent shaped nuclei were seen in the upper layers of stratum spinosum and granulosum. Hyperplasia of basal cell layer was noticed with hyperchromatic nuclei and moderate mitotic activity. Dermis showed mild to moderate hyperplasia of the connective tissue that consisted of blood vessels, fibroblasts, focal hemorrhages and mild infiltration of lymphocytes. Other types (fibroma and fibrosarcoma) were characterized by proliferation of fibrous connective tissues with fibrocytes and fibroblasts of variable sizes arranged in different directions with pleomorphic and hyperchromatic nuclei and occasionally presence of mitotic figures. These CWs were tested for the presence of DNA of BPVs (BPV-1,-2,-5&-10) by Polymerase Chain Reaction and confirmed by sequencing (KF148686, HG007968, HG007969, HG007970, KF114853). PCR results revealed that 6 cases of cattle CWs were positive for BPV-1 and 2 for BPV-2, while 5 cases were positive for mixed infections of BPV-1 and-2 and one case each positive for BPV-2 & -5 and BPV-1, -2 & -5. In Buffalo CWs, BPV-1 was detected in 4 cases and 1 case each revealed the mixed infections of BPV-1 &-2 and BPV-1& 5. A total of 12 (8 cattle and 4 buffalo CWs) samples were negative for DNA of BPV-1,-2, &-5. All the samples were negative for BPV 10. The immunohistochemical expression of Ki-67 was restricted to nucleus only. The immunopositivity was predominantly observed in proliferating cells stratum spinosum while discrete immunopositive cells were cells were also present in basal layers in papilloma and fibropapilloma. Few cells in fibrous stroma also showed positive reaction. The immunoreactivity with intense nuclear staining was observed in occasional cases of fibrosarcoma. In conclusion, CWs in cattle and buffaloes are more frequently associated with BPV-1 & -2 and their mixed infections in India; with rarely presence of BPV-5 also. In fact, more frequent suprabasal Ki-67 expression suggest that it may have association with viral replication as it expressed in the spinosum layer where viral replication and assembly occurs.