https://www.bijenhouders.nl/files/Bijengezondheid/graaf/3.-avb-art-genersch-2009.pdf

American Foulbrood in honeybees and its causative agent, Paenibacillus larvae

A polyphasic taxonomic study of the two subspecies of Paenibacillus larvae, Paenibacillus larvae subsp. larvae and Paenibacillus larvae subsp. pulvifaciens, supported the reclassification of the subspecies into one species, Paenibacillus larvae, without subspecies separation. Our conclusions are based on the analysis of six reference strains of P. larvae subsp. pulvifaciens and three reference strains and 44 field isolates of P. larvae. subsp. larvae. The latter originated from brood or honey of clinically diseased honey bee colonies or from honey of both clinically diseased and asymptomatic colonies from Sweden, Finland and Germany. Colony and spore morphology, as well as the metabolism of mannitol and salicin, did not allow a clear identification of the two subspecies and SDS-PAGE of whole-cell proteins did not support the subspecies differentiation. For genomic fingerprinting, repetitive element-PCR fingerprinting using ERIC primers and PFGE of bacterial DNA were performed. The latter method is a high-resolution DNA fingerprinting method proven to be superior to most other methods for biochemical and molecular typing and has not previously been used to characterize P. larvae. ERIC-PCR identified four different genotypes, while PFGE revealed two main clusters. One cluster included most of the P. larvae subsp. larvae field isolates, as well as all P. larvae subsp. pulvifaciens reference strains. The other cluster comprised the pigmented variants of P. larvae subsp. larvae. 16S rRNA gene sequences were determined for some strains. Finally, exposure bioassays demonstrated that reference strains of P. larvae subsp. pulvifaciens were pathogenic for honey bee larvae, producing symptoms similar to reference strains of P. larvae subsp. larvae. In comparison with the type strain for P. larvae subsp. larvae, ATCC 9545T, the P. larvae subsp. pulvifaciens strains tested were even more virulent, since they showed a shorter LT100. An emended description of the species is given.

Reclassification of Paenibacillus larvae subsp. pulvifaciens and Paenibacillus larvae subsp. larvae as Paenibacillus larvae without subspecies differentiation

We evaluated the antagonistic effects of newly identified lactic acid bacteria (LAB) in the genera Lactobacillus and Bifidobacterium, originating from the honey stomach, on the honey bee pathogen, Paenibacillus larvae. We used inhibition assays on agar plates and honey bee larval bioassays to investigate the effects of honey bee LAB on P. larvae growth in vitro and on AFB infection in vivo. The individual LAB phylotypes showed different inhibition properties against P. larvae growth on agar plates, whereas a combination of all eleven LAB phylotypes resulted in a total inhibition (no visible growth) of P. larvae. Adding the LAB mixture to the larval food significantly reduced the number of AFB infected larvae in exposure bioassays. The results demonstrate that honey bee specific LAB possess beneficial properties for honey bee health. Possible benefits to honey bee health by enhancing growth of LAB or by applying LAB to honey bee colonies should be further investigated.

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Novel lactic acid bacteria inhibiting Paenibacillus larvae in honey bee larvae

American foulbrood (AFB) is a severe bacterial brood disease of honey bees. AFB is lethal to bee colonies if treatment is not carried out. The disease is distributed worldwide and can cause substantial economic losses. Foulbrood was first described in 1769 and although AFB has been in focus for decades it has recently become an increasing problem.

American foulbrood: a review of its biology, diagnosis and control

Summary American foulbrood is one of the most devastating diseases of the honey bee. It is caused by the spore-forming, Gram-positive rod-shaped bacterium Paenibacillus larvae. The recent updated genome assembly and annotation for this pathogen now permits in-depth molecular studies. In this paper, selected techniques and protocols for American foulbrood research are provided, mostly in a recipe-like format that permits easy implementation in the laboratory. Topics covered include: working with Paenibacillus larvae, basic microbiological techniques, experimental infection, and “’omics” and other sophisticated techniques. Further, this chapter covers other technical information including biosafety measures to guarantee the safe handling of this pathogen.

/pdf/standard-methods-for-american-foulbrood-research-s5q032h4tg.pdf

Standard methods for American foulbrood research.

SUMMARYThe sensitivity of five solid media for detection of Bacillus larvae spores in honey samples; J agar, MYPGP agar, brain heart infusion (BHIT) agar, Colombia agar and horse blood agar was compared in air or under 5% C02. MYPGP agar is superior to all other media tested for detection of low spore levels of B. larvae in honey. Incubation under 5% C02 enhances growth considerably, especially for MYPGP agar and J agar. The plating efficacy described here, where an average of 23% of inoculated spores germinated on MYPGP agar, is higher than reported elsewhere for B. larvae. Thus, contamination levels as low as 200 B. larvae spores/g of honey can be detected with more than 95% probability on MYPGP agar incubated under 5% C02 if 12 or more plates are inoculated.

Susann Nordström

Papers

A comparison of media and cultural conditions for identification of Bacillus larvae in honey