Rapid Detection of Actinobacillus pleuropneumoniae From Clinical Samples Using Recombinase Polymerase Amplification

Abstract

Actinobacillus pleuropneumoniae (APP) is the causative agent of porcine pleuropneumonia, resulting in high economic impact worldwide. There are currently 19 known serovars of APP, with different ones being predominant in specific geographic regions. Outbreaks of pleuropneumonia, characterized by sudden respiratory difficulties and high mortality, can occur when infected pigs are brought into naïve herds, or by those carrying different serovars. Good biosecurity measures include regular diagnostic testing for surveillance purposes. Current gold standard diagnostic techniques lack sensitivity (bacterial culture), require expensive thermocycling machinery (PCR) and are time consuming (culture and PCR). Here we describe the development of an isothermal point-of-care diagnostic test - utilizing recombinase polymerase amplification (RPA) for the detection of APP, targeting the species-specific apxIVA gene. Our APP-RPA diagnostic test achieved a sensitivity of 10 copies/μL using a strain of APP serovar 8, which is the most prevalent serovar in the UK. Additionally, our APP-RPA assay achieved a clinical sensitivity and specificity of 84.3 and 100%, respectively, across 61 extracted clinical samples obtained from farms located in England and Portugal. Using a small subset (n = 14) of the lung tissue samples, we achieved a clinical sensitivity and specificity of 76.9 and 100%, respectively) using lung imprints made on FTA cards tested directly in the APP-RPA reaction. Our results demonstrate that our APP-RPA assay enables a suitable rapid and sensitive screening tool for this important veterinary pathogen.

Laboratory tools for the direct detection of bacterial respiratory infections and antimicrobial resistance: a scoping review

“…These studies reported using ONT, 31 LAMP, 41 and RPA ( n = 2). 130 , 147 A rapid, long-read metagenomic sequencing study reported using ONT to detect bacterial and fungal pathogens in human, ventilator-associated pneumonia cases compared to a combined standard of culture and qPCR for pathogen detection. 31 They also compared metagenomic ARG detection to culture-based antimicrobial susceptibility testing and found high consistency between the ARGs detected from gram-negative bacterial pathogens with aminoglycoside and beta-lactam resistance phenotypes.…”

“…The only animal ONT study included in our scoping review investigated M. bovis in pooled respiratory samples of cattle. 16 The wide application of isothermal techniques for the direct detection of bacterial respiratory pathogens and ARGs in various human and animal settings could be because of the simplicity of operation, potential for a relatively short TAT, low LOD, and ease of interpretation of outputs (e.g., LAMP), 130,147,173 which make these attractive methods to monitor and control outbreaks in resource-limited settings. 41 We found that laboratory tests described as "rapid" had variable descriptions and inclusion of steps from sample collection to reporting of results.…”

The Evolution in Pharmacology of Infectious Diseases: 2022

“…pleuropneumoniae and P. multocida are one of the major contributors to porcine respiratory diseases worldwide. The symptoms of infection can include arthritis, breathing difficulties, and lethargy in young pigs (Jensen et al, 1999;Stringer et al, 2022). The incidence rates of A. pleuropneumoniae infection range from 25% to 48%, with a significant mortality rate, thereby causing significant economic losses in the industrial pig breeding industry (Vangroenweghe and Thas, 2021).…”

Optimizing tylosin dosage for co-infection of Actinobacillus pleuropneumoniae and Pasteurella multocida in pigs using pharmacokinetic/pharmacodynamic modeling

“…A. pleuropneumoniae and P. multocida are one of the major contributors to porcine respiratory diseases worldwide. The symptoms of infection can include arthritis, breathing difficulties, and lethargy in young pigs ( Jensen et al, 1999 ; Stringer et al, 2022 ). The incidence rates of A. pleuropneumoniae infection range from 25% to 48%, with a significant mortality rate, thereby causing significant economic losses in the industrial pig breeding industry ( Vangroenweghe and Thas, 2021 ).…”