A Sensitive and Accurate Recombinase Polymerase Amplification Assay for Detection of the Primary Bacterial Pathogens Causing Bovine Respiratory Disease.

Cheyenne C Conrad,Maurice Boissinot,Kingsley K Amoako,Shaun Cook,Trevor Alexander,Tim Mcallister,Brenda Ralston,Yan D Niu,Michel G Bergeron,Kim Stanford,Rahat Zaheer,Rana K Daher

doi:10.3389/fvets.2020.00208

Abstract

Rapid and accurate diagnosis of bovine respiratory disease (BRD) presents a substantial challenge to the North American cattle industry. Here we utilize recombinase polymerase amplification (RPA), a fast and sensitive isothermal DNA-based technology for the detection of four BRD pathogens (Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, Mycoplasma bovis), genes coding antimicrobial resistance (AMR) and integrative conjugative elements (ICE) which can harbor AMR genes. Eleven RPA assays were designed and validated including: a) one conventional species-specific multiplex assay targeting the 4 BRD pathogens, b) two species-specific real-time multiplex RPA assays targeting M. haemolytica/M. bovis and P. multocida/H. somni, respectively with a novel competitive internal amplification control, c) seven conventional assays targeting AMR genes (tetH, tetR, msrE, mphE, sul2, floR, erm42), and d) one real-time assay targeting ICE. Each real-time RPA assay was tested on 100 deep nasopharyngeal swabs (DNPS) collected from feedlot cattle previously assessed for targets using either culture methods and/or polymerase chain reaction (PCR) verification (TC-PCR). The developed RPA assays enabled sensitive and accurate identification of BRD agents and AMR/ICE genes directly from DNPS, in a shorter period than TC-PCR, showing considerable promise as a tool for point-of-care identification of BRD pathogens and antimicrobial resistance genes.

Highlights

Bovine respiratory disease (BRD) remains the most common and economically important disease affecting feedlot cattle, veal calves, weaned dairy heifers and beef calves [1, 2]
Using the TwistAmpTM Basic kit, recombinase polymerase amplification (RPA) assays were optimized for integrative conjugative elements (ICE) and each BRD species individually (M. haemolytica, P. multocida, H. somni, and M. bovis), as well as being used in a conventional multiplex containing all four BRD targets (Figure 3)
The real-time multiplex RPA assays are shown in Figures 4A,B, for P. multocida/H. somni and M. haemolytica/M. bovis, respectively

Summary

Introduction

Bovine respiratory disease (BRD) remains the most common and economically important disease affecting feedlot cattle, veal calves, weaned dairy heifers and beef calves [1, 2]. RPA for Detecting BRD Pathogens reported to be as high as 1 billion dollars annually, due to losses in production, increased labor expenses, drug costs, and death [5, 6]. BRD is characterized by complex interactions between the host’s immune system, bacterial (i.e., Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasma bovis) and viral (i.e., Bovine Herpes Virus-1, Parainfluenza-3, Bovine Viral Diarrhea Virus, Bovine Respiratory Syncytial Virus) pathogens and management practices that increase stress such as weaning and transportation [4, 6,7,8]. Suppression of the host immune system as a result of stress or viral infection can allow these pathogens to proliferate within the upper respiratory tract, spreading to the lower respiratory tract, resulting in lesions and acute pleuropneumonia [4, 6]

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Conclusion