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

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).

Mycoplasma bovis Infections—Occurrence, Diagnosis and Control

“…Traditionally, culture is used for the confirmation of BRD infections, but the incubation period for each examined bacterial pathogens is different and samples inoculated onto agar plates are often overgrown with other, fast growing bacteria. For that reason, the multiplex real-time PCRs used by the laboratories [ 49 , 50 , 76 ] are the most suitable for simultaneous direct detection of M. bovis and other pathogens involved in BRD, such as P. multocida , M. haemolytica and H. somni , in contrast to methods not dedicated for different pathogen identification in mixed infections such as one-target PCR, traditional culture or MALDI-TOF MS [ 77 ]. When using one target PCR, there is no information about the involvement of other pathogens in the disease, different bacteria have various growth requirements and slow growing bacteria can be easily overgrown by others, and MALDI-TOF MS is not able properly detect all organisms from polymicrobial samples.…”

Longitudinal study of humoral immunity to bovine coronavirus, virus shedding, and treatment for bovine respiratory disease in pre-weaned beef calves

“…DNA extracted from pooled nasal swab medium was sent to the University of Nebraska-Lincoln Veterinary Diagnostic Center for bacterial diagnostics by multiplex qPCR using the QuantiFast multiplex PCR kit (Qiagen Inc.) and primers and probes designed to detect M. haemolytica, P. multocida, H. somni and Mycoplasma bovis. This test has been validated for use with bovine nasal swabs and lung tissue matrices [24].…”

Evaluation of novel multiplex qPCR assays for diagnosis of pathogens associated with the bovine respiratory disease complex

“…These results suggest bacteria were present, but were undetected by culture possibly due to their fastidious nature or overgrowth by other organisms on culture (Bell et al, 2014;Van Driessche et al, 2017). PCR methods such as Pneumo4B are likely to overcome the effects of accompanying flora, and detect pathogens in mixed samples where traditional culture fails (Loy et al, 2018). The limitations of culture as a reference standard assay must be considered when determining the diagnostic specificity and sensitivity of Pneumo4B.…”

“…In other situations with multiagent diseases with opportunistic pathogens, quantification of the pathogens in clinical specimens has added substantially to the value of microbiological diagnosis (Lima et al, 2016;Pedersen et al, 2014Pedersen et al, , 2013. Recently, a multiplex quantitative PCR (qPCR) method to detect relevant bacterial pathogens of BRD was published, demonstrating better performance than culture in co-infections with two or more pathogens (Loy et al, 2018). To offer a quick and complete laboratory result of multiple BRD pathogens, a set of two commercially available multiplex qPCR assays has been developed under the name Pneumo4 (DNA Diagnostic A/S, Risskov, Denmark).…”