Microbial investigation on honey bee larvae showing atypical symptoms of European foulbrood

Culture-dependent and culture-independent techniques were applied to investigate the bacterial communities occurring in 5-6 days-old honey bee larvae (Apis mellifera L.), with evident symptoms of disease and healthy-looking ones. Samples were collected by the Bee Emergency Service Team (BeeNet Project) from two apiaries located in different geographical sites (North and South Italy). Observed symptoms were atypical, but very close to those attributed to the European foulbrood (EFB) and to the American foulbrood (AFB), which are severe diseases affecting honey bee larvae worldwide. Isolates from diseased larvae were identified as Enterococcus faecalis Schleifer et Kilpper-Balz and Paenibacillus dendritiformis Tcherpakov et al.. Melissococcus plutonius (ex White) Bailey et Collins corrig. Truper et de Clari, the causative agent of EFB, was detected by polymerase chain reaction in both diseased and healthy-looking larval samples, whereas Paenibacillus larvae White, the causative agent of the AFB, failed to be detected. Microbial profiles obtained by denaturing gradient gel electrophoresis did not show relevant differences among samples, thus evidencing that the healthy-looking samples were partially affected. Besides confirming the presence of E. faecalis and P. dendritiformis, as found by plate count, the technique confirmed the presence of M. plutonius in all samples. The study has evidenced that honey bee larvae were affected by the EFB, with the presence of an atypical Paenibacillus species as second invader, which presumably confers a different symptomatology to the diseased brood.