Reclassification of Paenibacillus larvae subsp. pulvifaciens and Paenibacillus larvae subsp. larvae as Paenibacillus larvae without subspecies differentiation Elke Genersch, 1 Eva Forsgren, 2 Jaana Pentika ¨ inen, 3 Ainura Ashiralieva, 1 Sandra Rauch, 1 Jochen Kilwinski 4 and Ingemar Fries 2 Correspondence Elke Genersch elke.genersch@rz.hu-berlin.de 1 Department of Molecular Microbiology, Institute for Bee Research, Friedrich-Engels-Str. 32, D-16540 Hohen Neuendorf, Germany 2 Department of Entomology, Swedish University of Agricultural Sciences, Box 7044, SE-75007 Uppsala, Sweden 3 National Veterinary and Food Research Institute, PL 92, FIN-70701 Kuopio, Finland 4 Staatliches Veterina ¨ runtersuchungsamt Arnsberg, Zur Taubeneiche 10-12, D-59821 Arnsberg, Germany 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 9545 T , the P. larvae subsp. pulvifaciens strains tested were even more virulent, since they showed a shorter LT 100 . An emended description of the species is given. INTRODUCTION American foulbrood (AFB), a fatal bacterial disease of honey bee brood, has spread worldwide (Matheson, 1993) and is a considerable threat to beekeeping in many countries as it is highly contagious and often lethal to managed bee colonies. The clinical symptoms of AFB are typical, with the brown, viscous larval remains forming a ropy thread when drawn out with a matchstick. The decaying brood desiccates into hard scales, tightly adhering to the walls of the cells, Abbreviations: AFB, American foulbrood; MRPs, macrorestriction profiles; rep-PCR, repetitive element-PCR fingerprinting. Published online ahead of print on 4 November 2005 as DOI 10.1099/ ijs.0.63928-0. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of Paenibacillus larvae strains 03-525, 00-1163, 02- 130 and 03-189 are DQ079620–DQ079623, respectively. A table giving the CCUG accession numbers for the P. larvae field strains used in the study and figures showing colony and spore morphology are available as supplementary material in IJSEM Online. 63928 G 2006 IUMS Printed in Great Britain 501 International Journal of Systematic and Evolutionary Microbiology (2006), 56, 501–511 DOI 10.1099/ijs.0.63928-0