Development and evaluation of real-time RT-PCR using ear hair for specific detection of sheep persistently infected with border disease virus (BDV)

Abstract

The aim of this study was to develop an improved border disease virus (BDV) specific real time RT-PCR and to evaluate its performance on manually plucked hairs from sheep persistently infected with BDV that may act as a non-invasive alternate sample. The BDV real time RT-PCR assay reported here showed a high analytical sensitivity (10^0.6 TCID50/ml), specificity (no reactivity with BVDV-1, BVDV-2, HoBi-like pestivirus and CSFV) and reproducibility. When the assay was validated on 210 samples from BDV-infected and uninfected sheep, it showed a 100% diagnostic sensitivity and specificity with virus isolation. Further evaluation of the assay on manually plucked hair follicles from ear (mid-lateral, mid-medial) and tail tip from sheep persistently infected with BDV showed that a minimum of 20 hair follicles need to be tested for correct diagnosis of BDV. The BDV load was comparatively higher in hairs from mid-medial ear than those from other tested locations. Evaluation on other samples from PI sheep demonstrated that the test performance was similar to that of pestivirus generic real-time RT-PCR, but improved than the currently available BDV specific real-time RT-PCR. Although more number of PI animals need to be evaluated, the results of the study showed that manually plucked hairs from mid-medial ear pinna is a suitable alternative sample in real-time RT-PCR for detection of BDV persistently infected sheep. Use of the non-invasive ear hair samples and the improved BDV specific real-time RT-PCR reported here may be useful for BDV surveillance in several sheep rearing countries.

Keywords: Border disease; Hair follicle; Persistent infection; Real time RT-PCR; Virus isolation.