The development of a loop-mediated isothermal amplification (LAMP) assay to detect American foulbrood in managed honey bee populations

Abstract

Paenibacillus larvae (American foulbrood) is a lethal and arguably the most destructive and economically important notifiable bacterial disease that severely impacts brood and colonies of the Apis mellifera (Western honey bee) worldwide. Detection in apiaries requires visual inspection of clinically symptomatic hives, which is unreliable, with laboratory confirmation required for definitive diagnosis. These methods can be costly, time-consuming, and require access to specialised equipment operated by experienced personnel. Disease confirmation is essential for notifiable diseases to mitigate spread and economic damages; therefore, rapid, sensitive, and specific point of care diagnostics are critical to prevent misdiagnosis and further outbreaks. To improve diagnostic turnaround, we developed a highly sensitive and specific novel loop-mediated isothermal amplification (LAMP) assay for the detection of P. larvae (AFB-LAMP), designed to amplify a small region of the DNA gyrase subunit B gene (GyrB) with 100% specificity demonstrated against non-target bacterial species of the honey bee gut microbiome and analytical sensitivity of 5 × 10⁻⁷ ng P. larvae with detection times within 20 min. To further reduce diagnostic resources and times, a bead-beating DNA extraction method suitable for field use was optimised which resulted in an AFB-LAMP diagnostic sensitivity and specificity of 97 and 98%, respectively. Thus, this AFB-LAMP is applicable for use in the field allowing for improved disease management of an agriculturally important species.