Loop-Mediated Isothermal Amplification for Detection of Pasteurella multocida, Mannheimia haemolytica, and Histophilus somni in Bovine Nasal Samples
This thesis aims to develop loop mediated isothermal amplification (LAMP) assays that can be used with bovine nasal samples to detect the presence of bacterial pathogens (Pasteurella multocida, Mannheimia haemolytica, Histophilus somni) that cause bovine respiratory disease. The most common method to diagnose and treat BRD involves a physical examination and follow-up trial-and-error antibiotic therapies. Unfortunately, physical symptoms are often not consistent with the presence of BRD and antibiotic treatments incur a failure rate of 33%. This can lead to a surgency of antibiotic resistant pathogens, posing a significant risk to the beef cattle industry. Nucleic acid-based diagnostics, such as polymerase chain reaction (PCR), offer a robust approach for identifying BRD pathogens by amplifying species-specific genes from genetic material present in nasal samples. However, PCR-based approaches are limited to a lab setting due to expensive equipment required for maintaining assay reactions, and inhibitors that necessitate preprocessing to optimize assay performance. LAMP, on the other hand, offers an accurate, inhibitor resistant approach to detecting BRD causing bacteria in a format more amenable to field use. This assay was developed to have an accuracy of 97% in pure DNA samples, sensitivity and specificity of 99% and 89% respectively in DNA-spiked bovine nasal samples, and has a limit of detection of 104 DNA copies/reaction.