11. Conclusions

The pathogenic mechanisms of EFB are poorly understood, and the factors and timescales leading to overt symptomatology remain enigmatic. Molecular tools will open new possibilities for the identification of putative virulence factors in both the bacterium as well as the host in order to unravel some of the pathogenic mechanisms. To date, there are no published methods for genotyping and molecular differentiation of M. plutonius strains, but the nucleotide sequence of the bacterial genome was recently deposited in the DNA Database of Japan under accession no. AP012200 and AP012201 (Okomura et al., 2011), and it is likely that new molecular methods such as genotyping will be developed in the near future. Moreover, research fields and methods already in use for research on P. larvae  such as selection of reference genes, quantifying and knocking down gene expression (see designated parts in the American foulbrood and molecular protocols papers of the BEEBOOK (de Graaf et al., 2013; Evans et al., 2013)) could be adapted to M. plutonius and EFB research. Moreover, new technologies may also be useful tools to study interactions between secondary bacteria and the causative agent and to fully understand their role in symptomatology.

Molecular diagnostic methods, such as PCR are also widely employed for EFB diagnosis. The PCR method is user friendly and theoretically, a single target DNA molecule is sufficient for detection, making it one of the most sensitive biological techniques ever described. Considering this, we might ask whether a positive PCR result is always biologically relevant. Low levels of M. plutonius can be found in apiaries where no symptoms of disease are present and the PCR will also detect non-viable bacterial cells. However, it is clear that M. plutonius is still below the level of detection in honey bee colonies located in some geographical areas (Budge et al., 2010). Future work should help understand whether this observation is due to the genetics of the honey bees from these areas, unfavourable meteorological conditions, lower apiary density, gut microbiota unfavourable to disease development, or simply down to an absence of movement of the causative organism.

Infectivity tests causing disease at the colony level using both cultured M. plutonius and extracts from diseased larvae were carried out during the 1930s (Tarr, 1936) and the 1960s (Bailey, 1960; Bailey, 1963; Bailey and Locher, 1968), but not much has been published since. This is an area of research where new information can be obtained by a combination of colony level infection experiments and modern diagnostic methods. Such advances would benefit from cross country collaborations, where advanced diagnostics from one country may complement field trials in another country where there may be less stringent rules governing EFB control.