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Another look at American Foul Brood

Dave Black

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The bacterial brood disease American Foul Brood (AFB) occurs worldwide and leads to significant losses of honey bee colonies every year. In several countries, as in New Zealand, AFB is a notifiable disease and infected bee colonies have to be burned to contain the disease. Although it has been under investigation now for more than a century, the underlying characteristics of the host–pathogen interactions on larval level remain elusive. An effective treatment of AFB does still not exist, partly since the progression of the disease following ingestion of spores has only been described superficially.


The bacteria that cause AFB belong to a large group of similar bacteria that can be both extremely useful and highly damaging. In 1988 scientists began to divide the main 'bacillus' classification into smaller, more comparable sub groups based on genetic, phylogenic principles rather than morphology, and one of the initial five sub-groups was 'Paenibacillus'. Literally, 'almost' bacillus. The species known for AFB became Paenibacillus larvae. The continual re-evaluation of the groups has led to the re-naming of some new species and the re-classification of existing species so that the classification continues to grow, and the genus currently has about 200 species. Paenibacillus is still expected to undergo significant taxonomic subdivision as these useful or otherwise relevant Paenibacillus continue to be studied, more are added, and the groups get unreasonably large.1-s2.0-S0022201109001864-gr4.jpg.fd6c64ef63d87da610093b2f0eff4c33.jpg


Many species of Paenibacillus produce antimicrobial compounds that are useful to fields like medicine, agriculture, industrial chemistry or bioremediation. Another well-known aspect of Paenibacillus is its role in the spoilage of milk and other dairy products, where its enzymes and metabolic products negatively impact the flavour or texture of dairy products, in, for example, the milk curdling caused by proteases. One of the challenges is that Paenibacillus are 'psychrotolerant' spore-forming bacteria. They form hardy spores, and function well at low temperature. Many Paenibacillus strains grow well in refrigerated temperatures used for storing milk. They represent more than 95% of bacteria in raw milk after 10 days of refrigerated shelf life, while spoilage of pasteurized milk due to Paenibacillus is delayed by the germination process of spores and usually occurs after 17–21 days. Paenibacillus species have also been used as pesticides to kill agricultural pests in a variety of ways. The chitinase enzymes they produce hydrolyse chitin, which is a structural polysaccharide of insect exoskeletons and gut linings. Like other bacteria, Paenibacillus species compete with other micro-organisms through the production of a wide range of antimicrobial compounds. All these general properties of Paenibacillus - curdling proteases, cool temperature range, chitin-destroying enzymes, persistent spores, and antimicrobial activity, are apparent in the symptoms produced by honey bee colonies infected by Paenibacillus.


For beekeepers, we know AFB is caused by strains of Paenibacillus larvae, each identity named after particular gene sequences known as ERIC (Enterobacterial Repetitive Intergenic Consensus) sequences. ERIC I and II are the types typically isolated from hives; ERIC III and IV are not considered to be significant. ERIC II is the most virulent strain, but ERIC I is more prevalent globally, not just because it can infect all honeybee subspecies, whereas ERIC II has been confined to to certain subspecies, but also because, being less virulent, it is more likely to be found. ERIC II will kill larvae in just 6 to 7 days. This strain produces a unique protein which facilitates attachment of the bacteria to part of the honeybee’s midgut. By contrast, ERIC I takes up to 12 days to kill infected larvae. The earlier larvae die the more effectively they can be removed as part of the social immune response of honey bees. By removing the dead larvae quickly during normal brood hygiene fewer bacterial spores are produced and spread in the hive. As a worker larval stage lasts six days ERIC I strains tend to die as pupae, while ERIC II strains can have deaths in open brood which are easily detected and removed by the bees. In one study only around 5% of ERIC II infected larvae died after cell capping, while 27% of the ERIC I infected larvae died after capping.

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Reports about the variable behaviour of AFB infections, and of the disappearance of infections in some circumstances, are presumably explained by the different expression of symptoms for each strain, and the variation between different strains and races of honey bee. For example, different species of honeybees produce subtly different royal jelly proteins, and this seems to affect their vulnerability to P. larvae infections. Apis mellifera ligustica’s royal jelly proteins are known to differ from those produced by Apis cerana. Interestingly, this difference in activity may provide Apis mellifera ligustica faster metabolic function and enhanced immune activity, and so the strain is a little more resistant to P.larvae (and A.apis) than is Apis cerana in that larval exposure to the spores results in infection less frequently. This may also help to explain the specificity observed in P. larvae ERIC II infections.  Until recently it had been assumed ERIC II was restricted to northern and central Europe, Argentina and Uruguay, but examples have now (2014) been isolated in Canada and New Zealand (Schäfer 2014).

Figure-1-Fluorescence-in-situ-hybridization-FISH-analysis-of-larval-sections-Larvae.thumb.jpg.e81284640362c533896a4d868cddeaa7.jpg

Honeybee larvae are most susceptible to infection by the bacteria's spores within the first 36 hours after egg hatching when larvae are fed with accidentally contaminated food. After metamorphosis bees are not susceptible to infection by the spores. As pupa they are not fed, and as adults they are able to void any spores they ingest. Foul brood eventually kills all the colony's offspring so there are no replacement workers to replace aging and dying individuals and the colony will collapse, often succumbing to robbers that spread the infectious spores in the process. Only a few spores are needed to initiate infection, but the number depends on the age, caste, and genetics of each larvae. As a rule, very young larvae are fed with brood food produced by nurse bee head glands, but as they age this food is progressively diluted by adding some of the honey or nectar content regurgitated by the nurses, increasing the risk of contamination. This is mitigated to an extent by the nurse's proventriculus filtering out spores, and with increasing age the larva's gut environment becomes progressively more hostile to the bacteria. By 48 hours after egg hatching most larvae are no longer vulnerable to the bacteria. The phospholipid lysophosphatidylcholine (LPC) has been identified as an antibacterial compound in honeybee midguts in both naturally developed and artificially reared honeybees. The concentration of LPC in royal jelly was below the limit of quantification, which probably indicates that it is not produced from the head glands. On the other hand, feeding experiments using fluorescent-labelled LPC indicate that it is delivered to young larvae with regurgitated honey stomach content to larvae. Fluorescence was found in middle-aged and older larvae to a higher extent than in young larvae, which corroborates that the delivery route via honey stomach becomes increasingly important with larval age. In older larvae and adults, in addition to a fully-formed peritrophic membrane, LPC is present in honeybee midguts, coexisting with the normal gut microbiota, as a permanently active immune trait representing a first line of defence against AFB and other infections. A healthy (lipid-rich) pollen supply is partly responsible for enabling LPC production.

59d7d339d1dca_Fig-5-Electron-micrograph-of-a-mature-endospore-of-Paenibacillus-larvae-MW-mothercopy.thumb.jpg.b246c97e0f75d5a84b533fd6a323efc0.jpg

Spores that have been ingested germinate rapidly in the midgut of infected larvae. Soon after ingestion vegetative bacteria can be found randomly distributed in the midgut cavity or 'lumen' and proliferate in the following days. Four to five days post infection the guts of larvae can be filled with P. larvae, while no bacteria are detected in the body, the midgut epithelium remains intact, and the 'bolus' containing the bacteria is still contained with the insect's peritrophic membrane, protecting the gut lining. This membrane, common to most insects, begins to form in larvae after about 8 hours and is thought to have evolved from the mucus lining the gut cells. It will in time form a barrier to protect the bees from mechanical abrasion and invasion by micro-organisms. As the bacteria multiply in the gut cavity they release compounds that have antibacterial and antifungal properties that kill any and all other species that might compete in the space. Metabolic fingerprinting (tracing carbon use) of P. larvae has revealed that this bacterium is able to use different sugars and sugar derivatives as a carbon source, so that the larval food and chitin containing larval structures can serve as food during this phase of infection. Both strains use chitinases that will degrade the peritrophic membrane for nourishment and once degraded they have access to the midgut lining. They eventually penetrate the midgut epithelium and migrate through the spaces between the cells into the haemocoel using an extensive arsenal of enzymes that destroy the proteins and collagen that bind the cells together, or bind the cells to extracellular matrices. At this point he host larvae dies and the toxins and enzymes produced continue to destroy and homogenise the body contents. When the larva dies a pure culture of P.larvae is present, a mix of both vegetative bacteria and bacterial spores. While we have always thought that the bacteria begins to form long-lived spores as a response to the immanent consumption of its  food supply it is now clear that spore formation begins immediately and continues through the entire infection process.

 

This article is available as a Portable Document (.pdf) here; The bacterial brood disease American Foul Brood.pdf


Photographs
1. An infected and normal pupa, cell cap removed, both 13 days old. The infected lava shows no body differentiation or segmentation and gravity makes it slump or ‘melt’ into the bottom of the cell. (Genersch 2010)
2. An advanced infection, with ragged and perforated and ‘wet’ cell caps, ooze through the cap, and scales forming on the cell floor. Note the brown colour of the exposed pupa. (Bee Informed Partnership)
3. Top/bottom row. Fluorescent markers added to the bacteria highlight the bacteria green against the yellow (or red) body parts. In B the large arrow points to a break in the gut wall. The images in the bottom row are of an uninfected larva. (Yue et al 2008)
4. Electron micrograph of a single P.larvae spore. The germ cell of the spore itself starts at position ‘GW’. The outer coats make it extremely resistant to desiccation, direct heat, chemicals, and antibiotics. (de Guzman et al 2011)

 

References and Reading
Current knowledge and perspectives of Paenibacillus: a review, (2016) Grady et al. Microbial Cell Factories 15:203 DOI 10.1186/s12934-016-0603-7


Reclassification, genotypes and virulence of Paenibacillus larvae, the etiological agent of American foulbrood in honeybees - a review, (2006) Ainura Ashiralieva, Elke Genersch  Apidologie, 37 (4), pp.411-420.

 

Strain and Genotype-Specific Differences in Virulence of Paenibacillus larvae subsp. larvae, a Bacterial Pathogen Causing American Foulbrood Disease in Honeybees, (2005)  Elke Genersch, Ainura Ashiralieva, and Ingemar Fries Applied and Environmental Microbiology, Vol. 71, No. 11 p. 7551–7555. doi:10.1128/AEM.71.11.7551–7555.2005

 

Rapid identification of differentially virulent genotypes of Paenibacillus larvae, the causative organism of American foulbrood of honey bees, by whole cell MALDI-TOF mass spectrometry. (2014) Marc Oliver Schäfer, Elke Genersch, Anne Fünfhaus, Lena Popping, Noreen Formell, Barbara Bettin, Axel Karger. Veterinary Microbiology Volume 170, Issues 3–4, Pages 291-297 https://doi.org/10.1016/j.vetmic.2014.02.006

 

How to Kill the Honey Bee Larva: Genomic Potential and Virulence Mechanisms of Paenibacillus larvae, (2014) Djukic M, Brzuszkiewicz E, Funfhaus A, Voss J, Gollnow K, et al. PLoS ONE 9(3): e90914. doi:10.1371/journal.pone.0090914

 

Paenibacillus larvae Chitin-Degrading Protein PlCBP49 Is a Key Virulence Factor in American Foulbrood of Honey Bees, (2014) Garcia-Gonzalez E, Poppinga L, Funfhaus A, Hertlein G, Hedtke K, et al.

PLoS Pathog 10(7): e1004284. doi:10.1371/journal.ppat.1004284

 

Fluorescence in situ hybridization (FISH) analysis of the interactions between honeybee larvae and Paenibacillus larvae, the causative agent of American foulbrood of honeybees (Apis mellifera), (2008) Dominique Yue, Marcel Nordhoff, Lothar H. Wieler, and Elke Genersch.  Environmental Microbiology 10(6), 1612–1620  doi:10.1111/j.1462-2920.2008.01579.x

 

Lysophosphatidylcholine acts in the constitutive immune defence against American foulbrood in adult honeybees, (2016) Ulrike Riessberger-Gallé, Javier Hernández-López, Gerald Rechberger, Karl Crailsheim & Wolfgang Schuehly. www.nature.com/scientificreports/ Scientific Reports | 6:30699 | DOI: 10.1038/srep30699.

 

Honey bee age-dependent resistance against American foulbrood (2001)  Karl Crailsheim, Ulrike Riessberger-Galle. Apidologie 32 (2001) 91–103 91.

 

Response of in-vitro reared honey bee larvae to various doses of Paenibacillus larvae larvae spores, (1998) Camilla J. Brødsgaard, Wolfgang Ritter, Henrik Hansen  Apidologie, 29 (6), pp.569-578.

 

Involvement of secondary metabolites in the pathogenesis of the American foulbrood of honey bees caused by Paenibacillus larvae, (2015)  Sebastian Muller, Eva Garcia-Gonzalez, Elke Genersch and Roderich D. Sussmuth  The Royal Society of Chemistry Nat. Prod. Rep., 2015, 32, 765.

 

Radiation inactivation of Paenbacillus larvae and sterilization of American Foul Brood (AFB) infected hives using Co-60 gamma rays, (2011) De Guzman, Z.M., et al  Applied Radiation and Isotopes, 69 pp1374-1379  doi:10.1016/j.apradiso.2011.05.032

 

American Foulbrood in honeybees and its causative agent, Paenibacillus larvae, Elke Genersch, (2010) Journal of Invertebrate Pathology Volume 103, Supplement Pages S10-S19

 

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yesbut

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" Honeybee larvae are most susceptible to infection by the bacteria's spores within the first 36 hours after egg hatching when larvae are fed with accidentally contaminated food. After metamorphosis bees are not susceptible to infection by the spores. As pupa they are not fed, and as adults they are able to void any spores they ingest. "

 

This implies to me they are able to voluntarily void spores. I presume that they are voided in the normal course of defecation ?

Edited by yesbut

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True, and not their only defence. Distinct from larvae with a blind gut - open only at the 'in' end. Is defecation 'voluntary'?

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8 minutes ago, Dave Black said:

Is defecation 'voluntary'?

Sometimes not !

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Tuck up with a cup of tea, and your favourite biscuit, this is quite a read. 

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15 minutes ago, Josh said:

this is quite a read. 

What is ?

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