Symptoms of EFB may easily be confused with other diseases or abnormalities in the brood, making diagnosis difficult. The diagnosis in the field can be further verified by microscopic examination of brood smear preparations (see section 6; Hornitzky and Wilson, 1989; Hornitzky and Smith, 1998), and a field test kit (see section 7.2) for the detection of M. plutonius in larval extracts is also available (Tomkies et al., 2009). Analysing pooled samples of bees from the brood nest by PCR (see section 8) may be an alternative or complement to visual inspection (Roetschi et al., 2008), although false negatives may sometimes occur (Budge et al., 2010). Sensitive detection methods are required to ensure the absence of the bacterium from bee products and for the confirmation of the visual diagnosis made in the field or for research purposes. Pure isolates of M. plutonius may sometimes be desirable for various research purposes (see section 5). There are selective media for the cultivation of M. plutonius (Bailey, 1957; Bailey, 1983; Bailey and Collins, 1982; Hornitzky and Wilson, 1989; Hornitzky and Karlovskis, 1989); but to culture the bacterium can be difficult and there is some evidence that M. plutonius samples from different regions have a differential response to culturing (Allen and Ball, 1993; Arai et al., 2012).
Immunology-based tests such as enzyme linked immuno-sorbent assay (ELISA) (Pinnock and Featherstone, 1984) have been published and used for the detection and quantification of M. plutonius (see section 7.1), but DNA amplification using the polymerase chain reaction (PCR) provides lower thresholds of detection than ELISA, and has been successfully used for the detection of M. plutonius since the late 1990s (see section 8).
This paper therefore aims to present selected protocols useful for diagnosis and research on the honey bee brood disease European foulbrood.