Graduation date: 2008Bovine respiratory syncytial virus (BRSV), bovine viral diarrhea (BVD) and bovine parainfluenza virus Type 3 (BPI3) are ubiquitous respiratory pathogens of cattle, which contribute to causation of bovine respiratory disease complex. As these respiratory viral pathogens cause very similar clinical symptoms, differential diagnosis of the pathogens is required in one sample. Therefore, a single tube fluorogenic multiplex real time-PCR-based TaqMan assay was developed for simultaneous detection of BRSV and BPI3 from bovine respiratory samples. TaqMan-PCR was optimized to quantify the viruses using the BioRad iCycler IQ real-time PCR detection system and dual-labeled fluorogenic probes. Primers and probes for our study were designed from a 100% conserved region of BRSV N gene and from BPI3 NP gene using GenBank sequences such that they can be used to differentiate BRSV and BPI3 in one-tube in clinical respiratory samples obtained from bovine species. Once the technique was optimized with validated reference strains and field isolates, the assay was applied to routine diagnostic samples. Respiratory samples collected from 100 cattle across the state of Oregon were screened for BRSV and BPI3 viruses by real time-PCR and virus isolation. The monoplex real-time PCR identified 19 samples positive for BRSV, 11 for BPI-3 and 14 for BVD. The multiplex real-time PCR identified 18 samples positive for BRSV and 4 samples positive for BPI3 Among positive samples, 2 samples showed positive for both BRSV and BPI3. BVD was left out of the multiplex reaction due to primer interference. In conclusion, the multiplex real-time TaqMan-PCR described here for detection and quantitation of BRSV and BPI3 viruses has been shown to be sensitive and specific. Rapid turnaround time, reproducibility and ease of use make this technique a valuable diagnostic tool for detection of BRSV and BPI3 in individual or pooled respiratory samples.