RESUMO
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 (100.6 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.
