Optimization of Immunohistochemical and Fluorescent Antibody Techniques for Localization of Foot-and-Mouth Disease Virus in Animal Tissues

Abstract

Immunohistochemical (IHC) and fluorescent antibody (FA) techniques were optimized for the detection of Foot-and-mouth disease virus (FMDV) structural and nonstructural proteins in frozen and paraformaldehyde-fixed, paraffin-embedded (PFPE) tissues of bovine and porcine origin. Immunohistochemical localization of FMDV was compared with 7 detection systems, 8 primary antibodies, and 11 epitope retrieval techniques. All serotypes tested (O, A, Asia, C [cryosection]; O, A, Asia [PFPE]) were localized in association with mature vesicles. Multi-label FA was used in conjunction with IHC and conventional histopathology to characterize vesicle maturation in 4 steers and 2 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. Cells at the leading edge of vesicles were positive for FMDV antigens by IHC and FA. Cell marker profile of these cells by FA was consistent with keratinocytes (i.e., cytokeratin [CK]-positive, S100-negative, MHC-II-negative). In rare instances, CK-negative, MHC-II- positive, and FMDV-positive cells (presumptive dendritic cells or macrophages) were identified within dermis subjacent to vesicles.

Morphologic and phenotypic characteristics of myocarditis in two pigs infected by foot-and mouth disease virus strains of serotypes O or A

“…Cardiac tissue samples were harvested during necropsy examinations, and were split into aliquots that were either frozen on liquid nitrogen for virus isolation and qRT-PCR [[9]], embedded in optimal cutting medium (Sakura Finetek, CA) and frozen above a bath of liquid nitrogen for immunomicroscopy [[10]], or fixed in 10% neutral buffered formalin for routine histology. Detection of FMDV antigen and phenotypic characterization of inflammatory cell populations in cryosections was performed by multichannel immunofluorescence (MIF) as previously described [[9],[10]].…”

“…Detection of FMDV antigen and phenotypic characterization of inflammatory cell populations in cryosections was performed by multichannel immunofluorescence (MIF) as previously described [[9],[10]]. Slides were examined with a wide-field, epifluorescence microscope, and images were captured with a cooled, monochromatic digital camera.…”

Clinical and virological dynamics of a serotype O 2010 South East Asia lineage foot-and-mouth disease virus in sheep using natural and simulated natural inoculation and exposure systems

“…Tissue samples were screened for contents of FMDV RNA using qRT-PCR , and subsequent detection of antigen in cryosections was performed by immunohistochemistry (IHC) and multichannel immunoflourescense (MIF) as previously described (Arzt et al, 2009;Arzt et al, 2010). Detection of FMDV antigen was performed using mouse monoclonal antibodies against structural proteins (VP1; F1412SA (Yang et al, 2007b)) and nonstructural proteins (3D; F19-6(302) (Yang et al, 2007a)).…”

Marker vaccine potential of foot-and-mouth disease virus with large deletion in the non-structural proteins 3A and 3B

“…The infected cells were fixed with cold acetone:methanol (1:1) mixture for 20 min at room temperature, after 7e8 h of infection. After washing the fixed cells twice with PBS, affinity purified t3AB specific bovine antibody and rabbit anti-cow immunoglobulin/HRP conjugate (DAKO, Denmark) was used for detection as per the method described before [21,22]. For indirect-immunofluorescent assay, BHK-21 cells grown on glass coverslips were mock infected or either infected with wildtype or mutated FMDV serotype O for 7e8 h at 37 C. After this time point, cells were fixed with cold acetone:methanol (1:1), permeabilized with 0.1% Triton-X-100 and blocked with 3% BSA.…”