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European foulbrood (EFB) is an important infectious disease of honeybee larvae, but its pathogenic mechanisms are still poorly understood. The causative agent,  Melissococcus plutonius , is a fastidious organism, and microaerophilic to anaerobic conditions and the addition of potassium phosphate to culture media are required for growth. Although  M. plutonius  is believed to be remarkably homologous, in addition to  M. plutonius  isolates with typical cultural characteristics,  M. plutonius -like organisms, with characteristics seemingly different from those of typical  M. plutonius , have often been isolated from diseased larvae with clinical signs of EFB in Japan. Cultural and biochemical characterization of 14  M. plutonius  and 19  M. plutonius -like strain/isolates revealed that, unlike typical  M. plutonius  strain/isolates,  M. plutonius -like isolates were not fastidious, and the addition of potassium phosphate was not required for normal growth. Moreover, only  M. plutonius -like isolates, but not typical  M. plutonius  strain/isolates, grew anaerobically on sodium phosphate-supplemented medium and aerobically on some potassium salt-supplemented media, were positive for β-glucosidase activity, hydrolyzed esculin, and produced acid from L-arabinose, D-cellobiose, and salicin. Despite the phenotypic differences, 16S rRNA gene sequence analysis and DNA-DNA hybridization demonstrated that  M. plutonius -like organisms were taxonomically identical to  M. plutonius . However, by pulsed-field gel electrophoresis analysis, these typical and atypical ( M. plutonius -like) isolates were separately grouped into two genetically distinct clusters. Although  M. plutonius  is known to lose virulence quickly when cultured artificially, experimental infection of representative isolates showed that atypical  M. plutonius  maintained the ability to cause EFB in honeybee larvae even after cultured  in vitro  in laboratory media. Because the rapid decrease of virulence in cultured  M. plutonius  was a major impediment to elucidation of the pathogenesis of EFB, atypical  M. plutonius  discovered in this study will be a breakthrough in EFB research.

Diversity of Melissococcus plutonius from Honeybee Larvae in Japan and Experimental Reproduction of European Foulbrood with Cultured Atypical Isolates