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.

Bee parasite movie: Lotmaria passim strain 'BRL' by R. Schwarz

This is a short microscopy movie of parasites from the honey bee (Apis mellifera) known as Lotmaria passim.  These cells look so nice and clean because they are from a pure cell culture I grow.  Note the flagella which they use for motility, as it whips about and pulls the parasite through the media.  These guys are kind of squished on the slide, so they are having a bit of trouble moving (this way I could get a good, clear image of them).  Normally, they can move about quite quickly.

Honey Bee Disease Strikes in All Seasons

Agricultural Research Service scientists have found that two pathogens causing mysterious honey bee ailments are not just a problem in the spring, but might pose a threat year round. Entomologists Ryan Schwarz and Jay Evans at the ARS Bee Research Laboratory in Beltsville, Maryland, and their colleagues have shown that two species of bacteria, Spiroplasma melliferum and S. apis, are more common than thought and infect honey bees in places as diverse as Brazil and Maryland.

To read the whole story, click on the post title or link below:
USDA ARS Online Magazine Bee

Apis mellifera on Aster where Spiroplasma bacteria may be transmitted from one bee to another.
Image Credit: Peggy Gruber, USDA/ARS.

"Mystery Spores" in Bee Macerates

Once way apiculturists assess their bee colonies for the relatively common microsporidian gut parasite Nosema and the need to treat their apiary against this pathogen or not (with fumagillin), is by doing a Nosema spore count from macerated honey bee abdomens.  If you've done this and looked at enough bee macerate samples, you may have seen the occasional "Mystery Spore".  You may have felt pretty certain it wasn't Nosema, pollen, or bee cellular debris but beyond that you weren't quite sure what it was you were looking at.  Often, I've heard people use the term "fried egg" cells as a general descriptor for such mystery spores.

"Mystery Spores" can be an important issue for at least 3 reasons:  

1) Getting an accurate Nosema spore count for research or treatment purposes can potentially be wildly incorrect if you confuse the sometimes ubiquitous (e.g. see "Mystery Spores #1") mystery spores for Nosema.

2) If they are fungal spores, some species of Aspergillus mold can be pathogenic to bees including A. flavus, A. nomius and A. phoenicis (Foley et al. 2014).

3)  If they are yeast spores, yeast communities in honey bees are poorly characterized and are a potentially diverse group of organisms associated with bee colonies that could use more research.


Below are two examples of "Mystery Spores" from honey bees that colleagues have sent me this spring.

Mystery Spores #1:  (image credit: Jim Burritt)

Background on Mystery Spores #1
Geographic location:  Wisconsin, USA.
Sample preparation:  fresh and in buffer.
Tissue source:  midgut specifically (carefully dissected out).
Associated with winter collapsed colonies (no, not CCD just dead-outs).
Cultivation:  unsuccessful growth on Sab Dex medium.
Notes:  Very abundant in source bees.  Lots of variation in size of nucleus.  


Mystery Spores #2:  (image credit: Michael Peirson and Carlos Castillo)

Background on Mystery Spores #2
Geographic location:  Alberta, Canada.
Sample preparation:  ethanol preserved or fresh frozen then suspended in PBS buffer.
Tissue source:  whole bee macerate.
Other source:  similar looking organisms were seen in pollen patties.
Notes:  Associated with bees fed with pollen patties; colonies without pollen patties did not seem to have these organisms.  No association with colony collapses.

Have some insight or a guess?

If you have some insight you'd like to share that may help determine what these are, we would appreciate if you Post a Comment below or send me an e-mail through the "Have something you want to share here???" link.
 
My Two Cents
There are a couple of candidates you may want to consider:  fungi/yeasts, trypanosomatids, Malpighamoeba, gregarines (e.g. Apicystis).  Based on prevalence alone, the first two candidates, fungi/yeasts and trypanosomatids are the most likely "mystery spore" you will encounter.  None of the above are trypanosomatid spheroids, I guarantee you (please see my previous post describing these cool but potentially problematic critters).  So, my best guess is that both Mystery spore #1 and #2 are yeast (Candida spp., Rhodotorula spp. etc.) or mold (Aspergillus spp.) spores.  What if it's Beauveria spp., common entomopathogenic fungi used as biocontrol agents in some areas that the bees are picking up?  These can probably (theoretically) be putatively identified to species by cultivating them and/or by purification and DNA amplification using universal fungal rRNA primers to generate sequence data that can be compared to cataloged species in GenBank.

The last two candidates, amoeba and gregarines, are apparently quite rare.  However, this may be due in part to difficulty discerning them and so they are under-reported.  Malpighamoeba mellificae can only be reliably identified during dissection and examination of intact Malpighian tubules, which is the tissue they infest creating diagnostic impactions (see Evans and Schwarz 2011).  Once they are disrupted from the tubules, they simply look like the nondescript "fried egg" cell and can't be reliably identified by any honey bee pathologist I know of currently (please speak up if you are an amoeba expert and disagree!).  There is no molecular data for Malpighamoeba mellificae yet so until that time, molecular diagnostics of this species is unavailable.  Some diverse gregarine taxa have been described from honey bees historically, particularly in honey bee colonies living in tropical environments (see Evans and Schwarz 2011) but recently only cross infection of honey bees with the bumble bee gregarine Apicystis bombi, have been reported (see Plischuk et al. 2011 and Ravoet et al. 2013).

Finally, I want to mention that cells such as these may be environmentally acquired and not truly infectious to bees, thus they may simply be 'passing through' inertly.  Only indisputable evidence of replication in the bees or a clear cellular/molecular level pathology would confirm organisms are infectious and pathogenic to bees, respectively.

References
Evans JD and Schwarz RS. 2011. Bees brought to their knees: microbes affecting honey bee health. Trends in Microbiology 19:614-620.

Foley K, Fazio G, Jensen AB, and Hughes WOH. 2014. The distribution of Aspergillus spp. opportunistic parasites in hives and their pathogenicity to honey bees.  Veterinary Microbiology 169:203-210.

Plischuk S, Meeus I, Smagghe G, and Lange CE. 2011. Apicystis bombi (Apicomplexa: Neogregarinorida) parasitizing Apis mellifera and Bombus terrestris (Hymenoptera: Apidae) in Argentina. Environmental Microbiology Reports 3:565-568.

Ravoet J, Maharramov J, Meeus I, De Smet L, Wenseleers T, Smagghe G, and de Graaf DC. 2013. Comprehensive bee pathogen screening in Belgium reveals Crithidia mellificae as a new contributory factor to winter mortality. PLoS ONE 8.

Reclassifying honey bee Trypanosomatidae parasites: Crithidia mellificae and Lotmaria passim

Finally available!  The full taxonomic characterization of one of the most abundant yet little understood parasites in honey bees, the trypanosomatid Lotmaria passim.

If you've detected trypanosomatids in your honey bees, it is possible they are the long recognized species Crithidia mellificae, but evidence now shows they are most likely to be the newly discovered species Lotmaria passim.

The manuscript includes light microscopy images for identification.  Hint:  If you think you've been seeing yeast spores in your gut macerates, take a closer look at the abundant "spheroid" stage of these parasites (see Figure 6 in the manuscript)!

Morphology may be carefully used to preliminarily discern L. passim from C. mellificae in honey bees (see below), but genetic confirmation is required for firm diagnosis.

Morphology comparison of the two honey bee trypanosomatid parasites.  (credit: R.S. Schwarz and Bee Bugs Blog)

GENETIC RESOURCES
Here are links to the NCBI Taxonomy page for L. passim and C. mellificae, where all current genetic Entrez records can be found.  The Table below (Table S1 from Schwarz et al. 2015) lists confirmed, reference genetic material for the type species.

Characterization of Two Species of Trypanosomatidae from the Honey Bee Apis mellifera: Crithidia mellificae Langridge and McGhee, 1967 and Lotmaria passim n. gen., n. sp Schwarz Journal of Eukaryotic Microbiology Wiley Online Library