Upon visual inspection in the field, large pieces of symptomatic brood may be cut out and sent to the laboratory (a piece of 10 x 10 cm cuts through the metal wires of the brood frame) for further examination and confirmation of the diagnosis. Correct sampling of brood is important because even within the same brood frame, M. plutonius is mainly found in larvae with visual disease symptoms (Forsgren et al., 2005). Alternatively, and in cases of lower severity, diseased larvae can be smeared on a microscope slide and submitted to the laboratory (see section 6). M. plutonius can survive for over three years on such slide preparations (Bailey, 1960), and 6 years within Lateral Flow Devices (Budge, unpublished data), and so culturing often remains a viable option many years after diagnosis.
It is possible to identify the presence of M. plutonius in the absence of disease symptoms by collecting bulk samples of 100 larvae, taken at random from across the brood nest and subjecting the samples to qPCR (Budge et al., 2010; see also the molecular methods paper of the BEEBOOK (Evans et al., 2013)). This method provided robust quantification of M. plutonius and is a potentially useful tool to help predict the risk of a colony either prior to disease development or in the absence of an inspection to confirm disease (Fig. 3).
Fig. 3. Estimates of the log amount of M. plutonius (MNE) in samples of larvae (A) and adult bees (B) probability of the honey bee colony being symptomatic for EFB. Data from 2006 are from Budge et al. (2010), and those from 2007 previously unpublished findings from follow up work. The plots represent estimates and corresponding 95% confidence limits from a generalized linear model constructed using qPCR data (see Budge et al., 2010).