American Foulbrood: Pathology, Biology & Beekeeper-do-this-ology

The Impact of AFB Disease in Infected Colonies

The most serious of the honey bee diseases, American foulbrood (AFB) is a bacterial disease that infects brood. Forager (or robber) bees contract the disease from the environment (or infected hives), whereby they return the contaminated nectar and/or honey back to their hive for processing. As nurse bees feed young larvae, they introduce the AFB bacterial spores into the larval body tissues, weakening and killing them. It’s the larvae, not the adults who get infected.

For the beekeeper, encountering this spore-forming bacterium is a death sentence as there are no effective treatments available. The spores enable bacteria to go dormant in the face of stressors, and can resist antibiotics treatments, remaining viable for decades. To prevent it spreading to other colonies, hives & equipment are destroyed by burning or sterilised through gamma irradiation (if permitted in your area).

Enough doom and gloom, it’s time for the (micro)biology of Paenibacillus larvae.

Symptoms of American Foulbrood

When inspecting brood cells, one of the first signs is the appearance of punctured or sunken cells and dark-brown cappings on the sealed brood; observing the entire frame would show an irregular brood pattern due to a mix of infected and healthy brood. The infamous “ropey” appearance is evident when the beekeeper conducts a matchstick test on an infected cell: The sticky mass can be drawn several centimetres outwards with a matchstick, and this ropey characteristic cannot be recreated in uninfected brood. Once you see the two in the field, you’ll know what to look for.

Larvae in uncapped cells go from a healthy, pearly-white colour to an off-white tone and finally a dark brown corpse as the bacteria consumes its larval host. The now-dead larvae dry into a dark scale that adheres to the lower cell wall. As the number of dead larvae increase, it produces the distinct foul smell that gives the disease its name, “foulbrood” (a smell thought to be caused by the production of ketone and sulfur-containing compounds).

From a whole-hive perspective, the infected colonies become less active and weaker, to the point where robber bees invade and capitalize on the weakened hive¹. Evidence of robbing includes bees fighting inside the hive body and at its entrance, as well as the appearance of “shredded” honey comb, as the robber bees quickly gather resources and return to their colony.

Diagnosing American Foulbrood in the Apiary

A laboratory test is required to confirm an American foulbrood infection, since symptoms manifest similarly to European foulbrood (EFB) or sacbrood virus infections. The laboratory diagnoses involves sending brood and comb samples to a regional laboratory, such as the Department of Agriculture in the U.S.. In addition, there are number of non-technical field tests available that can be conducted at the apiary.

Ropiness test

The matchstick test does not actually require a matchstick, and can be completed with a toothpick, the stem of a leaf, or small twig:

1. Poke the suspected brood cell, but not hard enough to break the cell wall.

2. Pull the chosen instrument out slowly and observe the brown, gooey mass roping out of the cell for at least 1/2 inch.

   1. If the result is negative, then the pupal mass will not stick (pun intended) to the instrument and fall away as its being pulled.

UV-light Visualization

The dry scale of larvae killed by AFB glows under ultraviolet light. Simply shine a blacklight torch onto the brood cells and watch the infected comb glow green-blue in colour, like in the image below.

The Holst Milk Test

The Holst Milk Test is based on the finding that diseased larvae curdle milk, unlike healthy larvae or those infected with other diseases, nor various hive products. This is due to the high level of protein-breaking enzymes (proteases) that are produced within the dead larvae, making it a solid field test for American foulbrood (Nizar et al., 2015). There are many methods to create the testing reagents, and one such example is listed below:

1. Add 1 teaspoon of powdered milk to 100 ml water and shake/stir to dissolve.

2. Once dissolved, equally pour the mixture into two smaller containers.

3. Gather the suspected infected larvae and add it to one of small containers.

   1. The other container serves as a control, ensuring that any observations are due to the larvae and not the milk itself.

4. Place both containers in a warm place (warm enough to activate the proteases within the infected larvae).

5. If the results are positive, then the test tube will into a clear brown, whereas the control tube remains unchanged as a white, cloudy liquid.

Further information on the Holst test is available in this short video.

AFB Rapid Diagnostic Test Kit

As the name implies, these are small, commercial devices for the diagnoses of AFB, such as the Vita beehealth test kit (and share a similar design to the rapid antigen test kits from the COVID years). Example instructions involve mixing the dead pupae with a testing reagent and adding it to the sample well. Two lines at “C” and “T” indicate a positive result.

Are there Treatments for American Foulbrood?

There are no effective treatments for AFB due to the tenacity of its spores, and infected hives are (usually) destroyed by burning – the spores have a such high degree of resilience to destruction it’s remarkable (to do with their tight cell wall structures). Drug treatment suppresses development of the AFB pathogen, slowing but not stopping the infection. Additionally, antibiotic treatment of honey bee populations is problematic due to the emergence of antibiotic-resistant strains as well as local regulations, which often require beekeepers to discontinue antibiotic treatment for some period of time prior to honey harvests.

Research into alternative treatments for American foulbrood is ongoing, and is centered around three different themes:

• Selective breeding of honey bee stock that has an increased individual or “social immune response” (hygienic) behaviour against AFB (reviewed in Al Toufalia et al., 2018),
• Using beneficial bacteria to combat P. larvae (think probiotics, or viruses that infect bacteria [bacteriophages]) (reviewed in Jończyk-Matysiak et al., 2020),
• Using natural antibacterial substances such as propolis or essential oils (Pellegrini et al., 2017),
• Vaccinating bees against P. larvae (pilot study results available in Dickel et al., 2022).

These topics deserve an article of their own, and the interested reader is directed to the references listed above for further information.

The Microbiology of American Foulbrood Spores

Non-technical readers are welcome to skip this section.

Microscopically, Paenibacillus larvae is a rod-shaped, gram-positive bacteria that forms endospores; a “dormant” stage of its lifecycle that provides it with high resistance to environmental pressures and chemical treatments. These endospores enable P. larvae to remain viable for decades in contaminated equipment and hive products. Only high-frequency irradiation and extreme heat (such as from the burning hive) have been successful in eradicating the endospores (The former method is useful in the event beekeeping equipment is reused.)

There are four different genotypes of P. larvae, and these strains are determined on the basis of repetitive elements within their plasmids, designated as ERIC I to ERIC IV (Enterobacterial repetitive intergenic consensus [ERIC]; Genersch & Otten, 2003). Of the four, ERIC I was found to be the least virulent, taking 12-14 days to kill infected larva, compared to the 6-7 days of the other four. Research by Rauch & colleagues (2009) showed that the slower AFB phenotype, ERIC I to be more infectious overall, and hypothesized that a slower disease progression led to a decreased likelihood of nurse bees clearing out infected larvae before the bacteria had converted its host into a mass of infectious spores.

For the ERIC II phenotype, the infected larvae were efficiently removed prior to cell capping, disrupting disease spread and development, leading to less ropey, scale-containing cells. Don’t be mistaken, the colony will still collapse, however this will happen relatively slowly within the colony, compared to the fast colony collapse in ERIC I infections, giving beekeepers time to dispose of the infected hives and reducing the chances of transmission to other hives.

ERIC I & II are found in the field². ERIC II infected colonies display an orange pigmentation, and was historically misclassified as a subspecies of Paenibacillus larvae, P. l. pulvifaciens (Genersch et al., 2005). The non-pigmented ERIC I is the classic AFB infection seen in most images and media, causing the majority of worldwide AFB outbreaks; ERIC II is more common in Europe (Kilwinski et al., 2004).

Causes and Lifecycle

When the spores are ingested by larvae, they pass through the foregut into the midgut, commencing germination 12 hours after ingestion; the first vegetative spores are detectable within 24 hours (Yue et al., 2008). The bacteria begins colonizing the midgut and leeching the food ingested by its larval host, secreting active enzymes involved in the pentose-phosphate pathway and carbohydrate metabolism (reviewed in Genersch, 2009).

According to Yue et al., (2008), during the proliferative phase of P. larvae, (2-6 days post-infection), bacterial activity is restricted to the midgut. At later stages of the infection, the bacteria penetrates the peritrophic membrane and into the epithelium via phagocytosis, degrading its host into the dark brown hard mass characteristic of dead larvae. The result is the hard scale that adheres to the lower brood cell wall, containing millions of infectious spores. As nurse bees clear infected cells, they contaminate themselves with the vegetative spores, producing two routes of spore distribution: either infecting the next larva as they partake in brood rearing activities, or produce contaminated honey as they ripen nectar through trophallaxis.

Why are Adult Bees Unaffected by AFB?

As mentioned above, adult honey bees (and more recently, small hive beetles) are disease vectors. AFB spores germinate inside the larvae due to a variety of factors:

• Gut flora development: In the initial hours of a larva’s life, its gut is relatively free from (detectable) microorganisms (Martinson et al., 2012). As the larva is fed by the nurse bees, various bacteria establish themselves in the gut, making it harder for pathogens to dominate and cause infection. Unless of course, the larva is fed food contaminated with P. larvae.
• Immune system development: The innate immune system of honey bee larvae becomes more robust as they develop. Younger larvae are hence more susceptible to AFB infection compared to older larvae.
  • One form of this immune system is the development of the “Peritrophic matrix”, which surrounds the food within the larval gut, retaining the food (and by extension, P. larvae) within the gut area (Yue et al., 2008).
• Established microbiome of adult honey bees: adults have a core gut microbiome of nine bacterial phylotypes, whereas the larval microbiome is different and far more variable than the adult bees, despite the crossover in taxa (Kowallik & Mikheyev 2021).
  • At the beginning and end of pupation, large parts of the gut lining is shed or remodeled via ecydsis, eliminating the accumulated internal bacteria that the the larval stage (Zheng et al., 2018). Throughout pupation, there is an absence of gut bacteria, and the newly emerged worker bees will receive it via social transmission (such as trophallaxis and allogrooming) after emergence from their cells.

Where is American Foulbrood Found?

Geographically, American foulbrood is found all over the world and its global spread is attributed to human activities. As it stands, there is no definitive origin of AFB, and one possible hypothesis is that it co-evolved alongside honey bees in a pathogen-host relationship that eventually resulted in this specialized interaction. It wasn’t until the differentiation of AFB from EFB in 1906 that the name American foulbrood was designated, as it was first determined that the former Bacillus larvae (now Paenibacillus larvae) was responsible for causing the American foulbrood disease.

Check your local regulations. In many parts of the world, AFB is part of the agricultural department’s notifiable diseases list, and notifications of infections are required by law, with penalties for failing to do so. Better to lose your bees than to lose your bees and your money!

Extracting Honey from AFB Infected Hives

For the beekeeper, extracting the honey from an infected hive is a somewhat similar process to regular extractions with extra precautions; this honey should only be used for human consumption.

The extraction process should be completed in an enclosed area that will not attract forager bees, as they would rapidly return with their nest mates, and bring the AFB spores back to their hive. Any beekeeping equipment used in the extraction will be deemed contaminated (eg, uncapping fork/knife, uncapping tray, extractor, buckets), and would require sterilization (gamma-irradiation from a commercial operator is the gold standard, otherwise a strong bleach or lye [sodium hydroxide] could work, be sure to not get the chemicals on your skin!).

For the extracted frames, their fate is in the fire, as they should be burnt to reduce the risk of spread; the frames will contain residual honey, and that will attract honey bees. Any leftover wax (from cappings or otherwise) is discarded or rendered at high temperatures to kill the spores, and not reused as frame foundation. If the honey had made from chemical-free forage, then the extracted product can be deemed from organic honey bees.

Killing the Diseased Honey Bee Colonies

Ultimately, the fate of an infected colony is incineration. Dig a pit deep enough to place the sealed brood box (the supers can be removed, so long as they’re free of bees), and then set alight. Petrol can be used as a firestarter, and once it combusts, the wooden material will eventually become ashes, ready for disposal.

Not a pretty sight, nor a pretty thought, yet that is the nature of beekeeping and agriculture. The prevalence of AFB along with other environmental stressors has been attributed to the disappearing honey bees phenomenon, and ongoing work in areas like P. larvae vaccination shows potential in reducing the impact of American foulbrood disease.

Comparing American Foulbrood with American Foulbrood

We conclude with a table. For further reading on EFB, refer to the article on European Foulbrood.

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Footnotes

1. A quick fix for robber bees is to reduce the size of the hive entrance to assist the infected hive in guarding. The issue with robber bees is that they spread the AFB spores to other colonies as they steal contaminated honey from the weak colonies, hence the importance of robbing prevention. ↩︎
2. The authors suggest that phenotypes ERIC III & IV went extinct as they killed larvae at such a rapid rate that the worker bees were able to clear out infected cells prior to bacteria reaching its spore-forming phase, and thus the high virulence of these two phenotypes were their downfall at the colony level. ↩︎