Development of an ante-mortem diagnostic test for enzootic nasal tumor virus and detection of neutralizing antibodies in host serum.
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.
Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada.
Scott R
SR
Walsh
Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada.
Kevin J
KJ
Stinson
Department of Population Medicine, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada.
Paula I
PI
Menzies
Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada.
Sarah K
SK
Wootton
D000818:Animals; D057134:Antibodies, Neutralizing / Q000097:blood*; D000914:Antibodies, Viral / Q000097:blood; D030121:Betaretrovirus / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D003584:Cytological Techniques / Q000379:methods; D003955:Diagnostic Tests, Routine / Q000379:methods*; D015511:Goat Diseases / Q000175:diagnosis*; D006041:Goats; D009656:North America; D012192:Retroviridae Infections / Q000175:diagnosis / Q000662:veterinary*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D012698:Serologic Tests / Q000379:methods; D012756:Sheep; D012757:Sheep Diseases / Q000175:diagnosis*; D014412:Tumor Virus Infections / Q000175:diagnosis / Q000662:veterinary*
D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral