9.2. Protocol for inducing EFB infection in honey bee larvae reared in vitro
A protocol for inducing EFB involves grafting an individual larva (less than 24 hours old) into a single well in a micro-titer plate (for detailed protocols see the in vitro rearing paper of the BEEBOOK (Crailsheim et al., 2013). Older larvae may also become infected but are less susceptible. Each larva is fed 10 µl of larval diet (Crailsheim et al., 2013) containing a defined number of M. plutonius cells (e.g. 500,000; see section 9.1.2). From 72 hours post grafting, the larvae are examined for mortality and fed uninfected feed daily, following the feeding regime recommended in Crailsheim et al. (2013). The mortality of the larvae can be evaluated using a microscope or by eye. Dead larvae are distinguished by the lack of respiration and loss of body elasticity.
The ability of M. plutonius to produce symptoms in the absence of secondary bacteria such as P. alvei seems to differ regionally. In Australia, feeding only M. plutonius has been demonstrated not to produce the typical clinical signs of EFB, but in Europe, M. plutonius was capable of inducing significant mortality in isolation (Charrière et al., 2011). When infecting with M. plutonius in combination with P. alvei, the larval colour changes to a greyish brown rather than a yellowish colour and the gut content of infected larvae turns watery rather than pasty. Infecting larvae with M. plutonius and subsequently feeding P. alvei (e.g. 60,000 spores in 10 µl larval diet) after 72 hours may produce signs typical of that seen in field cases of EFB (Giersch et al., 2010). The simultaneous or 3 days delayed inoculation of P. alvei has, however, been demonstrated not to influence the virulence of some European strains of M. plutonius (Charrière et al., 2011). The feeding of P. alvei in addition to M. plutonius has no influence on larval mortality as such, but may be important for the presence of all the typical EFB-symptoms, and the saprophyte P. alvei is probably important for the presence of some of the clinical symptoms in the field.