The Western honey bee ( Apis mellifera ) is among the most important commercial pollinators in agriculture, but also plays a central role as pollinator in natural ecosystems. The globally occurring brood disease American foulbrood (AFB), caused by the gram-positive, spore-forming bacterium Paenibacillus larvae , poses a serious threat to colony health and productivity. Early and accurate diagnosis is therefore essential to effectively contain disease outbreaks. This study aimed to develop a rapid, sensitive point-of-care test in lateral flow format for the simultaneous detection and differentiation of the clinically relevant P. larvae genotypes ERIC I and II. To achieve this aim, two target antigens were selected, produced recombinantly or purified, and used to generate monoclonal antibodies. These antibodies formed the basis for the development of two sandwich ELISAs and, building on this, a duplex lateral flow assay. Both immunoassay formats were evaluated with infected larvae samples. The results showed high specificity, sensitivity, and accuracy, allowing reliable detection of P. larvae and differentiation between the genotypes ERIC I and II, thereby providing a valuable addition to existing diagnostic methods. In particular, the lateral flow assay enables rapid on-site diagnosis, facilitating timely intervention, and thus supports effective measures to control the spread of the disease.
