Differentiation of Paenibacillus larvae subsp. larvae, the cause of american foulbrood of honeybees, by using PCR and restriction fragment analysis of genes encoding 16S rRNA

Abstract

A rapid procedure for the identification of <i>Paenibacillus larvae</i> subsp. <i>larvae</i>, the causal agent of American foulbrood (AFB) disease of honeybees (<i>Apis mellifera</i> L.), based on PCR and restriction fragment analysis of the 16S rRNA genes (rDNA) is described. Eighty-six bacterial strains belonging to 39 species of the genera <i>Paenibacillus</i>, <i>Bacillus</i>, <i>Brevibacillus</i>, and <i>Virgibacillus</i> were characterized. Amplified rDNA was digested with seven restriction endonucleases. The combined data from restriction analysis enabled us to distinguish 35 profiles. Cluster analysis revealed that <i>P. larvae</i> subsp. <i>larvae</i> and Paenibacillus larvae subsp. <i>pulvifaciens</i> formed a group with about 90% similarity; however, the <i>P. larvae</i> subsp. <i>larvae</i> restriction fragment length polymorphism pattern produced by endonuclease <i>Hae</i>III was found to be unique and distinguishable among other closely related bacteria. This pattern was associated with DNA extracted directly from honeybee brood samples showing positive AFB clinical signs that yielded the restriction profile characteristic of <i>P. larvae</i> subsp. <i>larvae</i>, while no amplification product was obtained from healthy larvae. The method described here is particularly useful because of the short time required to carry it out and because it allows the differentiation of <i>P. larvae</i> subsp. <i>larvae</i>-infected larvae from all other species found in apiarian sources.