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Development of an ante-mortem diagnostic test for enzootic nasal tumor virus and detection of neutralizing antibodies in host serum.

The Journal of general virology (2014-05-20)
Scott R Walsh, Kevin J Stinson, Paula I Menzies, Sarah K Wootton
ABSTRACT

Enzootic nasal adenocarcinoma (ENA) is a contagious neoplasm of the nasal mucosa of sheep and goats and is associated with enzootic nasal tumour virus (ENTV). As ENA is a common disease in North America and there are no vaccines against ENTV-1, diagnostic tests that can identify infected animals and assist with eradication and disease surveillance efforts are greatly needed. In this study, we endeavoured to develop a novel, non-invasive diagnostic tool that could be used not only to validate clinical signs of ENA but also to detect ENTV-1 infection prior to the onset of disease signs (i.e. pre-clinical diagnosis). Cytology, serology and reverse transcription (RT)-PCR-based diagnostic methods were investigated. Although the cytology-based assay was able to detect ENTV-1 infection in some animals, it had poor sensitivity and specificity and thus was not developed further as an ante-mortem diagnostic method. Three different assays, including ELISA, Western blotting and virus neutralization, were developed to detect the presence of ENTV-1-specific antibodies in sheep serum. Whilst a surprisingly large number of sheep mounted an antibody-mediated immune response against ENTV-1, and in some cases neutralizing, correlation with disease status was poor. In contrast, RT-PCR on RNA extracted from nasal swabs reliably detected exogenous ENTV-1 sequences, did not amplify endogenous ovine betaretroviral sequences, demonstrated high concordance with immunohistochemical staining for ENTV-1 envelope protein, and had perfect sensitivity and specificity. This report describes a practical and highly specific RT-PCR technique for the detection of clinical and pre-clinical ENA that may prove beneficial in future control or eradication programmes.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
IPTG, ≥99% (TLC), ≤0.1% Dioxane
Sigma-Aldrich
Isopropyl β-D-1-thiogalactopyranoside, ≥99% (TLC)
SAFC
Isopropyl β-D-1-thiogalactopyranoside
Sigma-Aldrich
Isopropyl β-D-thiogalactopyranoside solution, ReadyMade IPTG solution for Blue-white screening