Optimization of fluorescent antibody techniques for demonstration of foot-and-mouth disease virus in bovine tongue epithelium and dorsal soft palate

Abstract

Nucleic acid detection and virus isolation (VI) techniques are preferred for many applications in diagnostic virology, but these techniques are not enough to provide microscopic localization virus or viral antigen at cellular or at tissue level. Though the anti-FMDV fluorescent antibody technique (FAT) has been used as a tool for investigation of viral pathogenesis, they could also be useful as an ancillary diagnostic tool, particularly when necroptic tissues might be the only available samples. In this study FAT was optimised for the detection of FMDV structural proteins in frozen tissues of bovine origin. Tongue epithelium (TE) and dorsal soft palates (DSP) collected from FMDV suspected animals and later confirmed for FMDV positive by RT-LAMP and its serotype by mPCR were used. TE and DSP of apparently healthy bovine collected from slaughter house used as negative control, which was previously confirmed negative for FMDV by RT-LAMP and mPCR. Fluorescent antibody labelling of FMDV was performed in conjunction with labelling of cell markers like pancytokeratin/β-tubulin. Epithelial cells were identified with anti-pancytokeratin for DSP while β-tubulin for TE. As expected, FMDV antigen was predominantly colocalized in vesicle along with β-tubulin/pancytokeratin. Tissues (TE & DSP) from the FMDV positive animals incubated with isotype control antibodies have no corresponding FMDV specific signal. The FAT established in the current study could identify virus-positive cells within the TE and DSP. As a research tool, this technique could allow the precise localization of FMDV during various stages of infection.