Antimicrobial resistance (AMR) in bovine respiratory disease (BRD) is an emerging concern that may threaten both animal and public health. Rapid and accurate detection of AMR is essential for prudent drug therapy selection during BRD outbreaks. This study aimed to develop a multiplex quantitative real-time polymerase chain reaction assay (qPCR) to provide culture-independent information regarding the phenotypic AMR status of BRD cases and an alternative to the gold-standard, culture-dependent test. Bovine clinical samples (297 lung and 111 nasal) collected in Nebraska were subjected to qPCR quantification of macrolide (MAC) and tetracycline (TET) resistance genes and gold-standard determinations of AMR of BRD pathogens. Receiver operating characteristic curve analysis was used to classify AMR based on the qPCR results. For lung tissues, the qPCR method showed good agreement with the gold-standard test for both MACs and TETs, with a sensitivity of 67–81% and a specificity higher than 80%. For nasal swabs, qPCR results passed validation criteria only for TET resistance detection, with a sensitivity of 88%, a specificity of 80% and moderate agreement. The culture-independent assay developed here provides the potential for more rapid AMR characterization of BRD cases directly from clinical samples at equivalent accuracy and higher time efficiency compared with the gold-standard, culture-based test.

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne’s disease, a severe gastroenteritis of ruminants. This study developed a model cell culture system to rapidly screen MAP mutants with vaccine potential for apoptosis. Two wild-type strains, a transposon mutant, and two deletion mutant MAP strains (MOI of 10 with 1.2 × 10⁶ CFU) were tested in murine RAW 264.7 macrophages to determine if they induce apoptosis and/or necrosis. Both deletion mutants were previously shown to be attenuated and immunogenic in primary bovine macrophages. All strains had similar growth rates, but cell morphology indicated that both deletion mutants were elongated with cell wall bulging. Cell death kinetics were followed by a real-time cellular assay to measure luminescence (apoptosis) and fluorescence (necrosis). A 6 h infection period was the appropriate time to assess apoptosis that was followed by secondary necrosis. Apoptosis was also quantified via DAPI-stained nuclear morphology and validated via flow cytometry. The combined analysis confirmed the hypothesis that candidate vaccine deletion mutants are pro-apoptotic in RAW 264.7 cells. In conclusion, the increased apoptosis seen in the deletion mutants correlates with the attenuated phenotype and immunogenicity observed in bovine macrophages, a property associated with good vaccine candidates.

Bovine Viral Diarrhea Virus (BVDV) is a significant pathogen of cattle, causing substantial economic losses. Identifying persistently infected (PI) animals is crucial for BVD control and prevention, with pooled ear-notch tissue testing being a standard screening method. Digital PCR (dPCR) is an emerging diagnostic tool that provides absolute quantification of animal pathogens in low abundant targets. This study evaluates the effectiveness of dPCR for BVD surveillance using field submissions of pooled ear-notch samples, comparing it to real-time quantitative reverse transcription PCR (qRT-PCR). The study compared performance using 107 pools of ear notches (4853 sample animals), including 76 BVD-positive pools (3543 sample animals) and 31 BVD-negative pools (1310 sample animals). Pools included those that contained PI animals detected by IHC (n = 25) and those with only virus detection that were negative by IHC (n = 51). Performance metrics evaluated included analytical and diagnostic sensitivity and specificity, limit of detection (LOD) and test agreement with qRT-PCR. Results showed that dPCR exhibited enhanced sensitivity and lower LOD compared to qRT-PCR, detecting as low as 0.6 viral copies/μL. dPCR achieved 100 % sensitivity (76/76) and 96.77 % specificity (30/31) compared to qRT-PCR and no detection of common bovine pathogens. The comparison between qRT-PCR and dPCR using Cohen's kappa coefficient in pooled samples was 0.98, indicating almost perfect agreement. In pooled ear-notch samples with qRT-PCR quantification cycle (Cq) values ranging from 30 to 33.99, the viral load ranged from 4.75 to 194.78 viral copies/μL. The study suggests that dPCR is a sensitive and specific method for detecting BVD in pooled ear-notches.