Detectability of Melissococcus plutonius and Paenibacillus larvae DNA in beeswax following thermal processing

Commercial beeswax recycling commonly relies on thermal processing (121 °C) to eliminate honeybee pathogens. However, residual DNA from inactivated pathogens can persist, causing highly sensitive qPCR assays to detect non-viable targets and complicate molecular surveillance. This study evaluated the post-treatment detectability of Paenibacillus larvae (American foulbrood, AFB) and Melissococcus plutonius (European foulbrood, EFB) DNA in apiary-derived wax samples and cultured bacteria before and after a simulated industrial autoclave treatment. Autoclaving profoundly reduced DNA amplifiability across all targets. In wax samples, all baseline-positive AFB samples became undetectable (Cq > 40). Conversely, M. plutonius DNA remained detectable in 5 out of 12 baseline-positive wax samples (post-treatment Cq range: 23.40 to 38.91). In matrix-free bacterial suspensions, both pathogens remained detectable after autoclaving, though P. larvae exhibited a smaller thermal amplification delay (+4.59 cycles) than M. plutonius (+9.66 cycles). In conclusion, standard high-pressure steam sterilization significantly degrades, but does not eliminate, M. plutonius DNA within beeswax from clinically diseased apiaries, allowing target fragments to remain detectable via sensitive qPCR assays. While P. larvae DNA is rendered completely undetectable in apiary-derived wax matrices, M. plutonius DNA can variably persist post-treatment. This contrasts with matrix-free bacterial suspensions, where DNA from both pathogens remains detectable across initial dilution gradients. Consequently, utilizing wax from these infected apiaries for foundation manufacturing can introduce historical DNA signatures that remain amplifiable, potentially confounding downstream molecular surveillance for identification of healthy carrier hives that are at risk of developing, or spreading, disease.