European Foulbrood: Microscopy and Diagnostics

Brief background
Melissococcus plutonius is the bacteria that causes a disease of honey bee larvae known as "European foulbrood" (EFB).  Bee keepers who suspect EFB by clinical signs in the colony ('scales', spotty brood pattern, smell) may want confirmation to help them decide how to best manage and control a potential outbreak in their apiary.  Diagnostic confirmation of EFB has traditionally required laboratory microscopy, but more recent techniques can be faster or more informative.

Microscopy
Confirmation of this disease can be made with staining and light microscopy examination.  Figure 1A shows what M. plutonius looks like during light microscopy examination.  Sometimes, however, the infection with M. plutonius may be largely cleared by the time examination is performed, and replaced with the secondary pathogens (Brevibacillus laterosporus (Figure 1B) and Paenibacillus alvei (Figure 1C).  What this means is, even if M. plutonius is not observed during examination, observation of B. laterosporus and/or P. alvei are pretty good indicators that M. plutonius was there, or is still there but in smaller numbers, and are indicative of EFB disease.

(1A)

(1B)

(1C)

Figure 1.  Light microscopy examination of A. mellifera brood "scales".  A) Melissococcus plutonius.  B) Brevibacillus laterosporus spores (the large, football-like shapes abundant throughout the image). C) Paenibacillus alvei (clustered in the central region of the image).   Specimen preparation by Sam Abban of the USDA Beltsville Bee Diagnostics Lab.  Photo credits: R. Schwarz @Bee Bugs.

Diagnostics
Other diagnostic measures can be performed to confirm EFB (M. plutonius) infection without microscopy and can be very sensitive detection options.  This includes detection of M. plutonius DNA using the molecular biology technique polymerase chain reaction (PCR).  This approach requires expensive equipment and reagents and is most useful for research purposes, offering the possibility of identifying genetic differences in EFB isolates (strains).  Alternatively, a 'snap test' is now available as a fast and easy alternative for simple presence / absence confirmation (Figure 2).

Figure 2.  European Foulbrood 'snap test'.  Photo credit: R. Schwarz Bee Bugs.

These 'snap tests'  are commonly used in veterinary medicine for parasite and pathogen diagnostics.  Briefly, the scale of interest (dead larva) is picked out of the comb cell, dropped into a lysis buffer, homogenized with metal beads, then a few drops are applied to the test membrane and you wait patiently for a few minutes.  A positive reference reaction will appear in the "Control" area on the membrane as a blue stripe, so you know the test is working properly (Figure 3).  If M. plutonius bacteria is present in your sample, a similar blue stripe will appear in the "Test" area of the membrane.

Figure 3.  Close-up of the European Foulbrood 'snap test' membrane.  "Control" area is on the right and the "Test" area is on the left.  This sample was positive for M. plutonius, confirming the microscopy examination shown above.

If there is no M. plutonius present, no color change will occur in this area of the membrane.  Simple as that!  If you want the ability to quickly confirm the presence/absence of EFB for management decisions, these snap test kits may be just what you're looking for.