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

  • Evangelia D. Apostolidi
  • Dimitra Psalla
  • Taxiarchis Chassalevris
  • Serafeim C. Chaintoutis
  • Nektarios D. Giadinis
  • Vassilios Psychas
  • Chrysostomos I. DovasEmail author
Original Article


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.



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.

Compliance with ethical standards


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

Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

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


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Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2019

Authors and Affiliations

  • Evangelia D. Apostolidi
    • 1
  • Dimitra Psalla
    • 1
  • Taxiarchis Chassalevris
    • 2
  • Serafeim C. Chaintoutis
    • 2
  • Nektarios D. Giadinis
    • 3
  • Vassilios Psychas
    • 1
  • Chrysostomos I. Dovas
    • 2
    Email author
  1. 1.Laboratory of Pathology, School of Veterinary Medicine, Faculty of Health SciencesAristotle University of Thessaloniki, University CampusThessalonikiGreece
  2. 2.Diagnostic Laboratory, School of Veterinary Medicine, Faculty of Health SciencesAristotle University of ThessalonikiThessalonikiGreece
  3. 3.Clinic of Farm Animals, School of Veterinary Medicine, Faculty of Health SciencesAristotle University of ThessalonikiThessalonikiGreece

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