title: Identification of Paenibacillus larvae to the subspecies level: an obstacle for AFB diagnosis.
abstract: This study was initially aimed at developing a PCR-test to differentiate between the pathogenic agent of American foulbrood (Paenibacillus larvae subsp. larvae) and powdery-scale disease (P. larvae subsp. pulvifaciens) of the honeybee. The test was based on the "insert of clone 9" (iC9), referring to a cloned 1.9 kB HaeIII fragment that occurs only in the P. larvae subsp. larvae reference strains and possibly correlates with American foulbrood virulence. It was shown that an iC9-based PCR-test discriminates between the BCCM/LMG reference strains of both subspecies. However, the screening of 179 Belgian field strains revealed five isolates that gave no iC9-based amplicon, thus rather resembling to P. larvae subsp. pulvifaciens. In addition, they all produced acid from mannitol, a characteristic previously assigned to the pulvifaciens subspecies. Because the reference strains gave conflicting data, this carbohydrate acidification was not conclusive. Therefore, the exact taxonomic position of the five retained strains was determined by a polyphasic approach using SDS-PAGE, AFLP, and ERIC-based PCR. Four iC9-negative field strains could be identified as P. larvae subsp. larvae; the taxonomic position of the fifth field strain remained ambiguous. The latter was provisionally classified as a subspecies pulvifaciens strain on the basis of SDS-PAGE. The present paper demonstrates the existence of field strains that do not fit well in the subdivision of the species P. larvae into two subspecies. Knowing that only one of both subspecies represents the pathogenic agent of AFB, this is a serious obstacle for the diagnosis of this honeybee disease.
authors.affiliation: Laboratory of Zoophysiology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium. Dirk.deGraf@UGent.be
authors.forename: Dirk C
authors.initials: DC
authors.lastname: de Graaf
authors.forename: Paul
authors.initials: P
authors.lastname: De Vos
authors.forename: Marc
authors.initials: M
authors.lastname: Heyndrickx
authors.forename: Stefanie
authors.initials: S
authors.lastname: Van Trappen
authors.forename: Nico
authors.initials: N
authors.lastname: Peiren
authors.forename: Frans J
authors.initials: FJ
authors.lastname: Jacobs
mesh_terms: D000818:Animals; D001407:Bacillus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001516:Bees / Q000382:microbiology*; D004269:DNA, Bacterial / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D020459:Genes, rRNA; D016937:Gram-Positive Endospore-Forming Rods / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008353:Mannitol / Q000378:metabolism; D010641:Phenotype; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length
chemical_list: D004269:DNA, Bacterial; D008353:Mannitol