“the failure gives you the opportunity to try a new approach”

Roger von Oech

I was very happy to attend a symposium focused on honeybee gut microbiome at Apimondia in Montreal. Topics included tolerance and detoxification of pesticides by honeybee gut microbiome members and impact on pests and pathogens of honeybees, from Nosema to Varroa. It was extended to additional symposia on honeybee health, which featured speakers on honeybee probiotics.

I did not expect to see studies of probiotics against American Foulbrood (AFB). Two studies summarized below tried to overcome AFB with probiotics, arriving at different conclusions. Comparing the two gives the reader a chance to compare scope of scientific inquiry and different methods of probiotic use.

Many researchers criticize the use of “generic” probiotic strains in honeybees. A team of microbiologists at Lund University have patented the product made entirely of honeybee-specific lactic acid bacteria, SymBeeotic.

SymBeeotic Ingredients: Lactobacillus kunkeei Fhon2N, Lactobacillus apinorum Fhon13N, Lactobacillus mellis Hon2N, Lactobacillus mellifer Bin4N, Lactobacillus apis Hma11N, Lactobacillus helsingborgensis Bma5N, Lactobacillus melliventris Hma8N, Lactobacillus kimbladii Hma2N, Lactobacillus kullabergensis Biut2N, Bifidobacterium asteroides Bin2N, Bifidobacterium asteroides Bin7N, Bifidobacterium asteroides Hma3N, and Bifidobacterium coryneforme Bma6N

In the study, Stephan et al. took 40 colonies started from packages in Beltsville, MD, USA, placed in an apiary with a history of AFB, and monitored for AFB symptoms¹. Frames and boxes came from colonies that were managed with AFB. Additionally, more AFB spores were fed to all colonies on day 9.

The colonies received were assigned randomly to groups receiving one of the following:

• antibiotic (tylosin) on day 21,
• placebo on days 1-3 and days 16-18,
• SymBeeotic on days 1-3 and days 16-18,
• no treatment (control)

The colonies were sampled and assessed for AFB symptoms over period of over 3 months and each colony was sampled six times during this period. This is incredibly methodical, detailed, and carefully analyzed study.

For such a thorough and labor-intensive study SymBeeotic application was not very well thought-out: mixed with honey and warm (45°C) sugar syrup, to less than 400,000CFU/g concentration, mixture was kept between 30 and 40°C overnight, and only 73ml of this mixture per colony was fed. This is a very low starting concentration and it’s questionable whether any bacteria remain viable after the long time before it was delivered. Probiotics work best when beneficial bacteria concentrations are high, 1,000,000,000 CFU/g or more.

Researchers found that only Tylosin suppressed clinical symptoms, while SymBeeotic group had same AFB scores as colonies receiving no treatment. Importantly, Tylosin did not lower AFB spores in the colonies, and Tylosin had additional negative effects on the colonies, Tylosin treated colonies suffered decreased colony size (frames of bees) and smaller brood size.

Canadian researcher Brendan Daisley and colleagues in London, Ontario, Canada, were testing new honeybee probiotic – BioPatty backed by California company Seed Labs². 6 colonies in a single apiary were assigned randomly to groups receiving one of the following:

• no treatment (control)
• patty without LAB on days 0 and 7
• patty with LAB on days 0 and 7

250-g patty contained standard pollen substitute ingredients (28.5 g of soy flour, 74.1 g of granulated sucrose, 15.4 g of debittered brewer’s yeast, 132.1 g of a 2:1 (w/v) simple sucrose-based syrup solution).

LAB: Lactobacillus plantarum Lp39, Lactobacillus rhamnosus GR-1, Lactobacillus kunkeei BR-1.

Unintentionally, Daisley’s apiary suffered exposure to AFB. The colonies were sampled and assessed on day 0 and 12 and euthanized and burned on day 12. All colonies showed AFB symptoms. Unfortunately, clinical symptoms were not scored. Short duration of the study and small group size must be noted.

Despite this limitation, probiotic bacteria were applied in a more reliable way on a patty. Importantly, lactic acid bacteria were applied at a final concentration of 1,000,000,000CFU/g.

Daisley found that 2 colonies that received LAB had significantly lower AFB spore counts. The group is continuing with more studies of their formulation, finding the results promising. Why did they see more promising results, at least with regard to AFB spore counts, than the SymBeeotic group? Daisley writes: “that Stephan et al. administered their supplemental bacteria using a 15% sucrose solution vehicle—likely resulting in a stark reduction of bacterial cell viability given this medium is known to induce severe osmotic stress in lactic acid bacteria”.

There’s really not enough evidence to recommend using probiotic approach in an active AFB infection, but a consistent methodology in applying probiotic supplements in bees can yield important insights. The differences between these two research articles, and similar disagreement among beekeepers in the field on how to correctly apply shows that standardized application methods, and best practices for honeybee probiotics are needed. Probiotic applied dry is more likely to be stable and effective:

References

Daisley BA, Pitek AP, Chmiel JA, Al KF, Chernyshova AM, Faragalla KM, Burton JP, Thompson GJ, Reid G. 2019. Novel probiotic approach to counter Paenibacillus larvae infection in honey bees. ISME J 2019 Oct 29. doi: 10.1038/s41396-019-0541-6

Stephan JG, Lamei S, Pettis JS, Riesbeck K, deMiranda JR, Forsgren E. 2019. Honeybee-specific lactic acid bacterium supplements have no effect on American foulbrood-infected honeybee colonies. Appl Environ Microbiol 85:e00606-19. https://doi .org/10.1128/AEM.00606-19