Pathological studies and detection of different bovine papilloma virus types in buffalo cutaneous warts

* Correspondence: [email protected] Bovine papillomatosis (BP) is distributed worldwide among cattle but is relatively less common in buffaloes. Prevalence of this disease is established in buffaloes from India (1,2) and Italy (3) caused by bovine papilloma virus (BPV). BPV is a nonenveloped double stranded DNA virus with icosahedral symmetry that infects the cutaneous and mucosal epithelia inducing hyperplastic lesions. Viral capsid proteins production takes place only in productive infections. The virion particles assemble in the granular and cornified layers; mature virus particles are released on disintegration of dead squamous cells (4). So far, 13 different BPV types have been identified and characterized. These are classified as Deltapapillomavirus (BPV-1, -2, and -13), Epsilonpapillomavirus (BPV-5 and -8), and Xipapillomavirus (BPV-3, -4, -6, -9, -10, -11, and -12), with the exception of BPV-7, which belongs to an as yet unassigned papillomavirus genus (5–8). Papillomatosis in buffaloes was reviewed and it was opined that it is an almost unknown disease in buffaloes and is caused by BPV-1 and -2 or their mixed infections. Once infection of BPV establishes in buffaloes, it spread from buffalo to buffalo, with no intermediate involvement of cattle (2). Various markers are expressed during cell division, each with a specific function. Expression of Ki‐67 nuclear antigen in all phases of the cell-cycle except for G0 has been recently found to be the most reliable indicator of cellular proliferation (9). Keeping the above in mind, the present work was carried out with the objective to study the occurrence and association of different BPV types with cutaneous warts (CWs) in Indian buffaloes, and their pathomorphological assessment. CWs/tumor biopsies from buffaloes were collected from the Referral Veterinary Polyclinic IVRI, Izatnagar, Government Veterinary Hospitals, different villages around Bareilly, India, and two archived samples from West Bengal, India. Biopsies were collected after administration of local anesthesia around the growth, samples were preserved in 10% buffered formalin for histopathological studies, and a part of samples showing gross lesions were also stored in sterile vials at –20 °C for molecular studies. Detailed gross observations with specific locations of growths/ wart-like lesions were recorded. After proper fixation, the representative tissues were dehydrated in increasing grades of alcohol, cleared in xylene, and embedded in paraffin blocks. Sections with 4–5 μm thickness were cut and stained with hematoxylin and eosin (H&E) as per the conventional procedure. DNA was extracted from the tissue samples using the Genomic DNA Mini Kit as per the manufacturer’s Abstract: Bovine papillomatosis is a common viral disease of cattle but is relatively less common in buffaloes. Bovine papilloma virus (BPV) -1 and -2 are mostly associated with cutaneous warts in buffaloes. The aim of the present work was to study the pathomorphology and association of different BPV types in buffalo cutaneous warts. A total of 10 cases were included in the present study, which revealed that growths were either single or multiple, variable in shape and size, and present at different body sites. Histopathologically, these were categorized as papilloma, fibropapilloma, and fibroma. Polymerase chain reaction (PCR) revealed the presence of BPV-1, -2, and -5 DNA. Quantitative real-time PCR showed varying amounts of viral DNA copy numbers. On immunohistochemistry, nuclear Ki-67 expression was observed mainly in the cells of suprabasal layers. In conclusion, DNA of BPV-1, -2, and -5 was detected either alone or their mixed infections. Nuclear Ki-67 immunoreactivity in the suprabasal layer indicated that this is the cellular proliferation site.