The present study was aimed to establish a novel TaqMan real-time PCR (RTm-PCR) for detecting and typing bovine viral diarrhea virus (BVDV), and also to develop a diagnostic proto- col which simplifies sample collection and processing. Universal primers and TaqMan-MGB probes were designed from the known sequences of conserved 5′ - and 3′-untranslated regions (5’UTR, 3’UTR) of the NADL strain of BVDV. Prior to optimizing the assay, cDNAs were tran- scribed in vitro to make standard curves. The sensitivity, specificity and stability (reproducibility) were evaluated. The RTm-PCR was tested on the 312 feces specimens collected from persistently infected (PI) calves. The results showed the optimum conditions for RTm-PCR were 17.0 μmol/L primer, 7.5 μmol/L probe and 51.4°C annealing temperature. The established TaqMan RTm-PCR assay could specially detect BVDV without detecting any other viruses. Its detection limit was 1.55×100 copies/μL for viral RNA. It was 10000-fold higher than conventional PCR with excel- lent specificity and reproducibility. 312 samples were tested using this method and universal PCR from six dairy farms, respectively. Positive detections were found in 49 and 44 feces samples, respectively. The occurrence rate was 89.80%. In conclusion, the established TaqMan RTm-PCR could rapidly detect BVDV and effectively identify PI cattle. The detection limit of RTm-PCR was 1.55 copies/μL. It will be beneficial for enhancing diagnosis and therapy efficacy and reduce losses in cattle farms.
The present study investigated the expression of androgen receptor (AR) in neurons of the anterior pelvic ganglion (APG) and celiac-superior mesenteric ganglion (CSMG; ganglion not involved in the innervation of reproductive organs) in the male pig with quantitative real-time PCR (qPCR) and immunohistochemistry. qPCR investigations revealed that the level of AR gene expression in the APG tissue was approximately 2.5 times higher in the adult (180-day-old) than in the juvenile (7-day-old) boars. Furthermore, in both the adult and juvenile animals it was sig- nificantly higher in the APG than in CSMG tissue (42 and 85 times higher, respectively). Immu- nofluorescence results fully confirmed those obtained with qPCR. In the adult boars, nearly all adrenergic (DβH-positive) and the majority of non-adrenergic neurons in APG stained for AR. In the juvenile animals, about half of the adrenergic and non-adrenergic neurons were AR-posi- tive. In both the adult and juvenile animals, only solitary CSMG neurons stained for AR. The present results suggest that in the male pig, pelvic neurons should be considered as an element of highly testosterone-dependent autonomic circuits involved in the regulation of urogenital func- tion, and that their sensitization to androgens is a dynamic process, increasing during the prepu- bertal period.