The development of a point-of-care diagnostic for the porcine respiratory disease complex

Abstract

The porcine respiratory disease complex (PRDC) consists of bacterial and viral pathogens, which commonly cause pneumonia in pigs and result in high economic losses worldwide. The pathogens involved include Actinobacillus pleuropneumoniae (APP), Mycoplasma hyopneumoniae (MH), Pasteurella multocida (PM), Streptococcus suis (SS) and Glaesserella parasuis (GPS), Swine influenza, Porcine circovirus-2 and Betaarterivirus suid. This Thesis describes the development of a diagnostic platform that can be used pen-side to identify the main PRDC pathogens. Analysis of four isothermal amplification methods determined that loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) produced rapid and specific amplification. LAMP achieved detection limits of 80, 20, 8, 8 and 20 copies/μL in the detection of APP, MH, PM, SS and GPS, respectively. RPA achieved 10, 20, 80 and 10 copies/μL for APP, MH, PM, and SS, respectively. Furthermore, LAMP and RPA achieved clinical sensitivities of 80.39% and 84.31% (n=61), 71.05% and 78.95% (n=123), 80.0% and 53.33% (n=92), 45.24% and 40.48% (n=121), and 79.66% (n=111) detecting APP, MH, PM, SS and GPS, respectively. LAMP conditions were successfully optimised and lyophilised, enabling ambient storage. Lyophilised LAMP displayed a slightly delayed amplification, but increased sensitivity compared to freshly prepared reactions. Additionally, an equipment-limited tissue extraction protocol was developed, and although less genetic material was extracted compared to the gold standard method, it was significantly more rapid (5 vs 70 minutes). 10 clinical lung samples were compared in sample-to-result using fluorescence-, lateral flow-, and colorimetric-LAMP detection methods with equivalent results. The colourimetric-LAMP was developed into a PRDC panel, and its usability was well received in a demonstration performed to a group of veterinarians. Implementing a rapid diagnostic tool that can rapidly (<35 minutes) identify and aid in the diagnosis of these important swine pathogens will prove a powerful tool to veterinarians, ensuring appropriate and prompt treatment.