Optimization of an in situ hybridization technique for the detection of foot-and-mouth disease virus in bovine tissues using the digoxigenin system

Woodbury, E. L.; Ilott, Martin; Brown, Corrie C.; Salt, Jeremy

doi:10.1016/0166-0934(94)00153-8

Cited by 17 publications

(3 citation statements)

References 5 publications

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“…This is the first microscopic documentation of localization of FMDV to the respiratory tract of any aerogenously infected animal earlier than 24 hpa. Two previous works have demonstrated nasopharyngeal intraepithelial FMDV RNA in cattle by in situ hybridization at 5 days after contact exposure; 20,26 however, at this later phase of infection, it is impossible to separate primary aerogenous from secondary hematogenous infection. The exclusive infection of PALT FAE cells in the context of pan-respiratory exposure to virus suggests that there are specific, intrinsic qualities of these cells that make them highly susceptible to infection.…”

Section: Discussionmentioning

confidence: 99%

The Early Pathogenesis of Foot-and-Mouth Disease in Cattle After Aerosol Inoculation

2010

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To characterize the early events of foot-and-mouth disease virus (FMDV) infection in cattle subsequent to simulated natural exposure, 16 steers were aerosol inoculated with FMDV and euthanized at various times. Samples were collected from each steer antemortem (serum, nasal swabs, and oral swabs) and postmortem (up to 40 tissues per animal) and screened for FMDV by virus isolation and for FMDV RNA by real-time reverse transcription polymerase chain reaction. Tissues that tested positive for FMDV or viral RNA were examined by immunohistochemistry and multichannel immunofluorescence microscopy. In previremic steers, FMDV was most consistently localized to nasopharyngeal tissues, thereby indicating this region as the most important site of primary viral replication. The earliest site of microscopic localization of FMDV antigens was the lymphoid follicle-associated epithelium of the pharyngeal mucosa-associated lymphoid tissue of the nasopharynx at 6 hours postaerosolization. At early time points after aerosol inoculation, viral antigens colocalized with cytokeratin-positive pharyngeal epithelial cells; intraepithelial FMDV-negative, MHCII/CD11c-double-positive dendritic cells were present in close proximity to FMDV-positive cells. Onset of viremia coincided with marked increase of viral loads in pulmonary tissues and with substantial decrease of viral detection in nasopharyngeal tissues. These data indicate that subsequent to aerogenous exposure to FMDV, the temporally defined critical pathogenesis events involve (1) primary replication in epithelial cells of the pharyngeal mucosa-associated lymphoid tissue crypts and (2) subsequent widespread replication in pneumocytes in the lungs, which coincides with (3) the establishment of sustained viremia.

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Section: Discussionmentioning

confidence: 99%

The Early Pathogenesis of Foot-and-Mouth Disease in Cattle After Aerosol Inoculation

2010

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“…Paraformaldehyde (PFA) fixation has been used predominantly in research applications when less protein and nucleic acid cross-linking is desired; paraformaldehyde-periodate-lysine (PLP) 15 fixation was originally described for electron microscopy but has been reported to be a superior fixative for IHC techniques owing to its ability to use lower concentration of PFA and thus induce less cross-linking 13 . Detection of FMDV RNA by ISH has been reported in PFA-fixed 22,28 and NBF-fixed 32 tissues. Antigen retrieval techniques from aldehyde-fixed tissues vary widely according to target antigen and operator preference, but generally consist of heat-induced epitope retrieval (HIER), enzymatic antigen retrieval, or a combination of both 23 …”

Section: Introductionmentioning

confidence: 99%

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

et al. 2009

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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.

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“…A fact behind this is that the primers used in MRT-PCR had a melting temperature within the range of 57-65 0 C. Henegariu et al (1997) reported an inverse effect of annealing temperatures of above 60 0 C on amplification efficiency. It is not uncommon to have field samples containing a mixture of two or more serotypes as reported earlier (Hedger et al, 1972;Woodbury et al, 1995). Because of problems with multiple infected samples for laboratory diagnosis , the ability of mPCR to detect all the three serotypes in a sample mix was tested.…”

Section: Bpmentioning

confidence: 99%

Standardization of Multiplex Reverse Transcription-Polymerase Chain Reaction and Typing of Foot-and-Mouth Disease Virus Prevalent in Bangladesh

Zinnah

Islam

Rahman

et al. 2012

Bangl. J. Vet. Med.

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Foot-and-mouth disease (FMD) is a devastating viral disease of cattle that causes severe economic losses in terms of loss of production and calf mortality in Bangladesh. Despite of regular vaccination, outbreak of the disease has become a regular event throughout the country every year. Determination of prevailing serotypes of the causal agent foot-and-mouth disease virus (FMDV) is now crucial need for strategic vaccination programme. The present research work was aimed to standardize a multiplex RT-PCR assay typing of foot-and-mouth disease virus serotypes prevalent among cattle population of Bangladesh. Uniplex and multiplex RT-PCRs were successfully developed and standardized using the extracted RNA of reference FMDV (Type A, O and Asia 1) following adjustment of the concentration of the viral RNA of each serotype, volume of reaction mixture and thermal profile. The mPCR was evaluated on 82 field samples (vesicular fluid, tongue epithelium and tissue from inter-digital space) of the years 2007 and 2008. Of the 82 field samples, 56 (68.29%) were found positive for FMDV. The mPCR successfully differentiated single as well as dual serotypes infection. The serotypes A, O and Asia 1 were confirmed in the samples of the year 2007 and only serotype O in samples of the year 2008. Higher detection rate was found in vesicular fluid (100%) followed by tongue epithelium (79.66%). It may be concluded that the MRT-PCR standardized in this study could be used for detection and differentiation of FMDV serotypes using field samples.DOI = http://dx.doi.org/10.3329/bjvm.v8i2.11199 Bangl. J. Vet. Med. (2010). 8 (2) : 149-155

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Optimization of an in situ hybridization technique for the detection of foot-and-mouth disease virus in bovine tissues using the digoxigenin system

Cited by 17 publications

References 5 publications

The Early Pathogenesis of Foot-and-Mouth Disease in Cattle After Aerosol Inoculation

The Early Pathogenesis of Foot-and-Mouth Disease in Cattle After Aerosol Inoculation

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

Standardization of Multiplex Reverse Transcription-Polymerase Chain Reaction and Typing of Foot-and-Mouth Disease Virus Prevalent in Bangladesh

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