|GREGG, DOUGLAS - Retired ARS Employee|
|CLAVIJO, ALPHONSO - Animal And Plant Health Inspection Service (APHIS)|
Submitted to: Journal of Veterinary Diagnostic Investigation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/15/2009
Publication Date: 11/20/2009
Citation: Arzt, J., Gregg, D.A., Clavijo, A., Rodriguez, L.L. 2009. Optimization of immunohistochemical and fluorescent antibody techniques for localization of foot-and-mouth disease virus in animal tissues. Journal of Veterinary Diagnostic Investigation. 21(6):779-792.
Interpretive Summary: This manuscript describes the development of novel techniques for the microscopic localization of foot-and-mouth disease virus (FMDV) in animal tissues. The techniques are all based on the specificity of certain monoclonal antibodies for FMDV. Immunohistochemical techniques allow visualization of localized FMDV via a conventional light microscope whereas immunofluorescent techniques utilize a fluorescent microscope. These novel protocols were applied to tissues from cattle and pigs experimentally infected with FMDV. The results provide a unique description of the distribution of FMDV in animal tissues and a characterization of the infected cells. These techniques are valuable tools for improving understanding of the mechanisms of FMDV pathogenesis.
Technical Abstract: Immunohistochemical (IHC) and immunofluorescent (IF) techniques were optimized for the detection of foot-and-mouth disease virus (FMDV) structural and non-structural proteins in frozen and paraformaldehyde-fixed paraffin embedded (PFPE) tissues of bovine and porcine origin. Immunohistochemical localization of FMDV was compared with seven detection systems, eight primary antibodies, and eleven epitope retrieval techniques. All serotypes tested (O, A, Asia, C for cryosection / O, A, Asia for PFPE) were localized in association with mature vesicles. Double and triple label IF were used in conjunction with IHC and conventional histopathology to characterize vesicle maturation in four steers and two pigs experimentally infected with FMDV. At the edge of advancing vesicles, a consistent finding was acantholytic degeneration of basal keratinocytes surrounding dermal papillae with suprabasilar clefts and microvesiculation. Progression of microvesiculation led to coalescence with the expanding vesicle-proper. Cells at the leading edge of vesicles were positive for FMDV antigens by IHC and IF. Cell marker profile of these cells by IF was consistent with keratinocytes (i.e. cytokeratin (CK) - positive, S100 - negative, MHC-II –negative). Rarely, CK-negative, MHC-II positive, FMDV - positive cells (presumptive dendritic cells or macrophages), were identified within dermis subjacent to vesicles.