Development of a Multiplex Real-Time PCR Assay for Predicting Macrolide and Tetracycline Resistance Associated with Bacterial Pathogens of Bovine Respiratory Disease.

Enakshy Dutta,Emily L Wynn,John Dustin Loy,Caitlyn A Deal,Jennifer Clarke,Bing Wang,Michael L Clawson

doi:10.3390/pathogens10010064

Abstract

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.

Highlights

Bovine respiratory disease (BRD) is one of the most common and costly cattle diseases and affects 97% of feedlots, or 16% of cattle, with economic losses estimated to exceed 1 billion USD/year [1,2]
For the H. somni strains, 100% identity to ICEtetR gene targets was observed for sequences in GenBank; the remaining three targets were not found in H. somni strains available at the time of analysis
The sequence identity of the primers and probes with non-BRD pathogen bacterial species does not indicate off-target binding to other genes, as it is expected that these antimicrobial resistance (AMR) gene sequences are found in various other bacterial species

Summary

Introduction

Bovine respiratory disease (BRD) is one of the most common and costly cattle diseases and affects 97% of feedlots, or 16% of cattle, with economic losses estimated to exceed 1 billion USD/year [1,2]. Disease onset is commonly initiated by viral infections, which may suppress host defense mechanisms, allowing opportunistic bacterial pathogens to replicate and colonize deeper in the lung. Environmental factors such as crowding, poor ventilation, weather, and weaning increase stress and reduce host immunity [4]. The development of AMR could compromise the effectiveness of antibiotics for the treatment and control of BRD, potentially leading to higher mortality and lower productivity among cattle populations. Tools for rapidly and accurately detecting potential AMR are critical components of outbreak monitoring by veterinarians and producers Such tools could be used as adjuncts to existing culture-independent BRD pathogen detection methods that use similar technology and workflows [13]. The use of these tools would enable rapid evaluation of the potential efficacy of antimicrobial interventions and benefit the beef industry and support antimicrobial stewardship by enabling informed selection of optimal drugs for BRD therapies

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