Author(s): Mota, Renato Veloso ; Carvalho, Paulo Miguel Martins ; Rodrigues, Diana Alexandra Ferreira ; Ribeiro, Artur ; Santos, Sílvio Roberto Branco ; Oliveira, Ana
Date: 2024
Persistent ID: https://hdl.handle.net/1822/94377
Origin: RepositóriUM - Universidade do Minho
The dormant and resilient spore-form of the Gram-positive bacteria Paenibacillus larvae represents a significant biological threat to honeybee populations (crucial pollinators worldwide) causing American Foulbrood (AFB). Spores, the infection form of the disease, are easily spread across apiaries, and when germinate inside larval midgut causes degradation of the remaining body into a viscous and semi-fluid mass, the most common clinical symptom of the disease. As antibiotics are not an option due to public health concerns and strict legislation, the incineration of contaminated hives is mandatory, severely impacting the ecological balance and beekeeping economy. Clinical symptoms only emerge in larvae after disease outbreak and therefore, the early detection of spores in adult bees, in a sub- clinical stage, while no effects are noted in the hive, will enable beekeepers to timely implement sanitary protocols and strict biosecurity rules. Currently, methods for detecting P. larvae spores present significant drawbacks, including extended turnaround times of microbiological cultures or compromised accuracy in faster methods such as PCR and antibody-based assays (inhibitors, false positives, high sensitivity, high cost). To address this, we have developed a novel method for rapid and early detect P. larvae spores, combining upstream magnetic capture/purification through a spore-binding protein with downstream specific detection employing a P. larvae-specific bacteriophage-derived cell wall binding domain, PlyPl23_CBD. The findings revealed an efficiency of 90% spore capture from bee gut and a subsequent germination time of 3.5h. Then, specific detection under fluorescence microscopy was achieved through the PlyPl23_CBD fused with the Green Fluorescent Protein (GFP). Overall, the developed methodology enables the early detection of P. larvae spores, even during pre-clinical stages of infection, in less than 5h. Furthermore, it holds promising potential as the foundation for an in-situ detection kit, by replacing the GFP-provided fluorescent signal with a light or colorful indicator.
