Standardization of a quantitative multiplex real-time PCR system for the detection of bovine respiratory disease pathogens in clinical samples of dairy cattle from southern Brazil.

Publication date: Jul 23, 2026

Bovine respiratory disease (BRD) is an economically significant and diagnostically challenging syndrome affecting cattle worldwide. Molecular investigations of BRD in Brazil were based exclusively on single-plex assays. This study presents the development, standardization, and field application of a quantitative real-time PCR (qPCR) system designed for the detection of 10 agents of BRD in dairy cattle from Southern Brazil using a panel based multiplex qPCR methodology. The qPCR system targeted bovine coronavirus (BCoV), bovine respiratory syncytial virus (BRSV), bovine viral diarrhea virus (BVDV), bovine alphaherpesvirus 1, bovine parainfluenza virus 3, ovine gammaherpesvirus 2, Histophilus somni, Pasteurella multocida, Mannheimia haemolytica, and Mycoplasmopsis bovis. Data were analyzed using descriptive, univariate, and multivariate statistical methods. Associations between pathogen detection, clinical status, and epidemiological factors were assessed using Chi-square tests, Fisher’s exact tests, generalized linear models, permutational multivariate analysis of variance, and non-metric multidimensional scaling. Assay repeatability and reproducibility were evaluated using one-way ANOVA. Standard curve analysis demonstrated uniform and robust analytical performance across all assays, with slopes ranging from -3. 553 to -3. 176, coefficients of determination ≥0. 991, and efficiencies between 91. 19% and 106. 48%. Most pathogens were reliably detected at dilutions up to 1: 10,000 or 1:100,000, confirming high assay sensitivity. Field testing of 115 nasal swabs revealed that 70. 4% of cattle were positive for at least one pathogen, with singular detections being most common (43. 5%). H. somni was the most frequently identified agent (45. 2%), followed by P. multocida (30. 4%), BCoV (10. 4%), and BRSV (9. 6%). Singular, dual, triple, quintuple, and sextuple pathogen detection patterns were identified by the qPCR assay in animals of distinct age-related groups and clinical status. Pathogen detection was significantly associated with age, season, and farm-level factors. BVDV and BRSV varied by age-related group (P ≤ 0. 006) and season (P = 0. 001), while BCoV (P = 0. 041) and H. somni (P = 0. 014) showed seasonal associations; multivariate analysis confirmed significant effects of age (P = 0. 001), season (P = 0. 001), and farm (P = 0. 003) on overall pathogen occurrence. One-way ANOVA demonstrated excellent repeatability and reproducibility of the qPCR platform, with highly consistent Cq values across independent analyses, testing days, and assay formats. Comparable performance was observed between single- and multiplex assays, including duplex, triplex, and fourplex configurations, confirming the analytical stability and reliability of the assay (P > 0. 05). These results demonstrated that the qPCR assay can detect singular and multiple pathogens in animals of diverse age groups and with and without clinical manifestations of BRD. Collectively, this study provides the first validated multiplex qPCR platform for the detection of BRD pathogens in Brazil and demonstrates its utility for identifying pathogen patterns in symptomatic and asymptomatic cattle. This qPCR system enables the rapid and simultaneous detection of major BRD pathogens, will reduce the cost associated with pathogen detection, and will be fundamental to understand the pathogenesis and epidemiology of BRD in Brazil.

Semantics

Original Article