Development of a multiplex real-time PCR assay using two thermocycling platforms for detection of major bacterial pathogens associated with bovine respiratory disease complex from clinical samples

Authors: LOY, JOHN - UNIVERSITY OF NEBRASKA; LEGER, LAURA - UNIVERSITY OF NEBRASKA; Workman, Aspen; Clawson, Michael - Mike; BULUT, ECE - UNIVERSITY OF NEBRASKA; WANG, BING - UNIVERSITY OF NEBRASKA

Submitted to: Journal of Veterinary Diagnostic Investigation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/30/2018
Publication Date: 9/21/2018
Citation: Loy, J.D., Leger, L., Workman, A.M., Clawson, M.L., Bulut, E., Wang, B. 2018. Development of a multiplex real-time PCR assay using two thermocycling platforms for detection of major bacterial pathogens associated with bovine respiratory disease complex from clinical samples. Journal of Veterinary Diagnostic Investigation. 30(6):837-847. https://doi.org/10.1177/1040638718800170.
DOI: https://doi.org/10.1177/1040638718800170

Interpretive Summary: Bovine respiratory disease complex (BRDC) is one the most significant diseases of cattle and causes losses to the US cattle industry in excess of $1 billion dollars annually. Approximately 70% of cattle have lung lesions indicative of prior respiratory disease at slaughter and approximately 65% of those were never diagnosed or treated. This tremendous cost and high incidence of undiagnosed animals underscores the need for new rapid and accurate diagnostic tools to mitigate the impact of BRDC on beef cattle production and enable targeted prevention and treatment strategies. Thus, the purpose of this study was to develop a new assay to quickly and accurately detect BRDC bacterial pathogens in clinical samples. Implementation of this testing method by veterinary diagnostic labs could enhance detection of bacterial pathogens and provide more rapid results to enable targeted treatment strategies.

Technical Abstract: Bovine respiratory disease complex (BRDC) is one of the most significant diseases of cattle. Bacterial pathogens involved in BRDC include Mannheimia haemolytica, Mycoplasma bovis, Histophilus somni, and Pasteurella multocida. We developed and evaluated a multiplexed real-time hydrolysis probe (rtPCR) assay using block-based Peltier and rotary-based thermocycling on lung tissue, nasal swabs, and deep nasopharyngeal swabs. The rtPCR results were compared to culture or a gel-based M. bovis PCR using statistical analysis to determine optimum quantification cycle (Cq) cutoffs to maximize agreement. The limits of detection were 1.2–12 CFU/reaction for each pathogen. M. haemolytica was the most prevalent organism detected by rtPCR, and was most frequently found with P. multocida. The rtPCR assay enabled enhanced levels of detection over culture for all pathogens on both thermocycling platforms. The rotary-based thermocycler had significantly lower Cq cutoffs (35.2 vs. 39.7), which maximized agreement with gold standard culture or gel-based PCR results following receiver operating characteristic analysis to maximize sensitivity (Se) and specificity (Sp). However, overall assay Se and Sp were similar on both platforms (80.5% Se, 88.8% Sp vs. 80.1% Se, 88.3% Sp). Implementation of these tests could enhance the detection of these pathogens, and with high-throughput workflows could reduce assay time and provide more rapid results. The assays may be especially valuable in identifying coinfections, given that many more antemortem samples tested in our study were positive for 2 or more pathogens by rtPCR (n = 125) than were detected using culture alone (n = 25).