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Development of real-time PCR-based methods for the detection of enzootic nasal tumor virus 2 in goats

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Abstract

Enzootic nasal adenocarcinoma (ENA) is a contagious neoplasm of sheep and goats, associated with the oncogenic retroviruses enzootic nasal tumor virus (ENTV) 1 and 2, respectively. It appears to be common in countries with substantial small ruminant-production. ENA diagnosis in goats is based on autopsy and histopathology, and there is no real-time PCR method available for ENTV-2 detection. Here, a novel one-tube real-time RT-PCR (RT-qPCR) method for the detection and quantification of ENTV-2 in nasal swabs is presented. The method targets the env gene/U3 region. For the design of ENTV-2-specific oligonucleotides, molecular characterization of seven Greek ENTV-2 strains was performed. Phylogenetic analysis revealed three distinct phylogenetic clades of ENTV-2 that correlate with the country of sample collection. Evaluation of the analytical performance of the RT-qPCR revealed an amplification efficiency of 92.8% and a linear range of quantification between 2 × 108 and 2 × 102 RNA transcripts. Analysis of nasal swabs from 23 histopathologically confirmed, naturally occurring ENA cases via RT-qPCR yielded positive results. Moreover, modification of the method for use in a real-time PCR (qPCR) assay enables detection of proviral DNA in tumor specimens. Both methods are highly specific and can be used for the confirmation of ENA-suspected cases. Future applications could include ante-mortem diagnosis, verification of the ENTV-2-free status in animal trade, disease surveillance, and control programs.

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Acknowledgements

Dr. Ioannis Ioannou (Head of Pathology, Bacteriology and Parasitology Laboratory, Cyprus Veterinary Services) is gratefully acknowledged for providing tissue samples from naturally occurring ovine ENA cases.

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Authors and Affiliations

  1. Laboratory of Pathology, School of Veterinary Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, University Campus, 54124, Thessaloniki, Greece

    Evangelia D. Apostolidi, Dimitra Psalla & Vassilios Psychas

  2. Diagnostic Laboratory, School of Veterinary Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, 11 Stavrou Voutyra Str., 54627, Thessaloniki, Greece

    Taxiarchis Chassalevris, Serafeim C. Chaintoutis & Chrysostomos I. Dovas

  3. Clinic of Farm Animals, School of Veterinary Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, 11 Stavrou Voutyra Str., 54627, Thessaloniki, Greece

    Nektarios D. Giadinis

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  1. Evangelia D. Apostolidi
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  2. Dimitra Psalla
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  3. Taxiarchis Chassalevris
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  6. Vassilios Psychas
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  7. Chrysostomos I. Dovas
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Correspondence to Chrysostomos I. Dovas.

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Funding

Evangelia D. Apostolidi was supported by the Greek State Scholarships Foundation (IKY).

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The authors declare that they have no competing interests.

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All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

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Apostolidi, E.D., Psalla, D., Chassalevris, T. et al. Development of real-time PCR-based methods for the detection of enzootic nasal tumor virus 2 in goats. Arch Virol 164, 707–716 (2019). https://doi.org/10.1007/s00705-018-04138-0

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  • DOI: https://doi.org/10.1007/s00705-018-04138-0

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