Science

American Foulbrood (AFB) is caused by the bacterium Paenibacillus larvae and is the most destructive of bacterial diseases afflicting honey bee brood worldwide. The infection process is a vicious cycle beginning when P. larvae spores are introduced into young larvae food and ingested. Infection is most likely to occur up to 48 hours after introduction into a larval cell. The spores enter the midgut of the larvae and reproduce until the midgut lumen is filled. The larvae die when P. larvae becomes overcrowded and bursts from the epithelium of the midgut into the haemocoel. Proliferation of the bacteria is observed by transition from beautiful pearly white to discolored shades of brown larvae. After the infected immature bee has been capped, sporulation occurs creating “sleeper cell” style spores which lie dormant in larval food, soil and equipment for decades and return to a vegetative state when ideal germination conditions are met.

The decaying capped immature bee deteriorates to form a blackened “scale”. Each scale is capable of releasing over 2 billion spores. Only one spore is necessary to potentially cause a widespread contamination throughout the hive and eventually infect neighboring hives. Spores are translocated throughout the hive as worker bees clean the dead infected brood from the cells and ingest the spores. The vicious cycle comes full circle as the worker bees, who are generally resistant to P. larvae, transfer the bacteria to other workers such as nurse bees who feed the larvae. Drifting, robbing and foraging bees are modes of transport to spread the infection to other hives.

Signs and Symptoms

Examine brood comb for brood pattern, larvae appearance and presence of black scale to check for possible AFB infection. Less common observations include seeing the pupal tongue protruding from scales. In advanced stages of the infection, a characteristic “foul-fishy” odor may be observed in addition to darkened, greasy and sunken capped cells. A test to determine AFB is called the “ropiness” test. This easy test uses a small straight object such as a stick and is inserted into an infected brood cell and agitated slightly. If AFB is present, slow withdrawal of the object from the cell will reveal a ropy glue-like consistency of vegetative P. larvae cells. If uncertain, collected brood samples can be sent to local and state apiarist agencies and laboratories. Apiarist laboratories use a variety of molecular biological testing techniques to diagnose an AFB infection.

When in Doubt, Send it Out!

Contact your local or state apiarist agency for lab testing.

Bee Research Laboratory: Beltsville, MD offers free of charge AFB diagnostic services

Lab testing will assess for AFB using some of the following techniques:

• Staining and Microscopy: The infected sample is mixed with water and a smear is placed on a cover glass. After heat fixing and staining the sample with carbol-fuchsin, the cover glass is inverted onto a microscope slide coated with immersion oil. The spore walls will appear reddish-purple with a clear center. Another technique uses a prepared slide including an infected bacterial sample mixed with fluorescently stained specific antibodies. Using a fluorescent microscope, larvae will appear fluorescent against a dark background.
• Inoculation: The infected sample is mixed in sterile water and heat shocked to kill any non-spore forming bacteria. A lawn of the sample is spread onto a plate containing yeast extract, soluble starch and glucose media and incubated for 72 hours at 34°C. Colony forming units will appear small and opaque. To confirm analysis, a catalase test is performed by adding a small amount of hydrogen peroxide to a colony on the plate. P. larvae will not form bubbles in the presence of hydrogen peroxide.
• Honey Analysis: Honey is diluted in water and centrifuged. The sediment is then examined using techniques mentioned in staining and microscopy. Another technique is to heat honey to 25°C, dilute with sterile water and run through a dialysis machine. After about 18 hours the sample is centrifuged and the sediment is isolated, diluted, heat shocked and then plated similar to technique mentioned in the inoculation section.

Prevention and Management

There are no medications, treatments or cures for AFB. AFB spores can lie dormant in hive equipment for decades withstanding high temperatures and freezing conditions.

Antibiotic treatments including Terramycin and Tylosin will target the bacteria but will not eliminate spores. These antibiotics are a band-aid option in that they only mask the symptoms and must be used for the entirety of the life of your hive. Avoid antibiotics at all costs to avoid developing antibiotic resistant strains of AFB. In addition, antibiotics and antimicrobials weaken the native hive microbial community, leading to microbial imbalances, weakened immunity and sterility.

Prevention Tips

• Inspect hive equipment regularly, be careful and critical when purchasing used equipment.
• Never move contaminated equipment into healthy hives.
• Never feed infected honey to a healthy hive.
• Burn contaminated equipment, especially frames, as necessary to avoid contaminating future hives.
• Clean hive tools after hive inspections with isopropyl alcohol, acetone or flame.
• If working in an area with a known AFB contamination, wash clothing and gloves in a weak bleach solution.
• Provide the highest quality nutrition and probiotic supplements like Super DFM Honeybee to aid in nutrient assimilation, prevent disease, stimulate immune defenses, antagonize pathogenic microbes and inhibit growth of bacterial diseases like AFB.

Prevention is the best option to reduce the likelihood of introducing a widespread AFB infection into your hives. Aside from good hive management practices, providing your bees with the highest quality nutrition and probiotic supplements will aid in the defense against harmful pathogens like P. larvae. Strong Microbials Inc. in Milwaukee, Wisconsin has been the first and foremost manufacturer of probiotic supplements for honeybees called SuperDFM Honeybee and contains over 75% of the naturally occurring microbes found in the honeybee gut such as lactobacillus and bifidobacterium.

References & Links

Dadant & Sons, Inc., The Hive and the Honey Bee, Hamilton Illinois, 835-836 p.

Grady, E; MacDonald, J; Liu, L; Richman, A; Yuan, Z. 2016. Current knowledge and perspectives of Paenibacillus: a review. Microbial Cell Factories: 15: 203. doi:  [10.1186/s12934-016-0603-7]