Development of a sandwich ELISA kit for detection of P. larvae and differentiation between its two main ERIC-genotypes

Honey bees are important pollinators that suffer from several diseases. The most devastating brood disease is American foulbrood, caused by Paenibacillus larvae, a bacterium with two main genotypes, ERIC I and ERIC II. Since these differ significantly in virulence, genotype differentiation enables the establishment of genotype-specific control measures and disease prevention. Therefore, an easy-applicable, fast and cheap laboratory diagnostic test is needed, especially for laboratories without the capability to perform PCR analysis. This study focused on the development of sandwich ELISAs that detect P. larvae in suspected larvae/pupae samples and are able to distinguish between the P. larvae with genotypes ERIC I and ERIC II. We generated monoclonal antibodies (mAbs) that enable the specific detection of P. larvae of both genotypes (limit of detection (LOD) > 3.8 × 102 CFU/mL), with 100% accuracy in in vitro-infected 5- and 6-day-old larvae compared with PCR detection, and also allow discrimination of P. larvae with the ERIC II genotype (LOD > 3.1 × 102 CFU/mL). A new sandwich ELISA kit was developed for the rapid detection of P. larvae with genotype differentiation. A storage stability test showed that its components, plates coated with mAbs and the ELISA reagents, are stable for at least 11 months stored at 4 °C.