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.

Paenibacillus larvae is the etiological agent of American foulbrood (AFB) in honeybees. Recently, different genotypes of P. larvae (ERIC I to ERIC IV) were defined, and it was shown that these genotypes differ inter alia in their virulence on the larval level. On the colony level, bees mitigate AFB through the hygienic behavior of nurse bees. Therefore, we investigated how the hygienic behavior shapes P. larvae virulence on the colony level. Our results indicate that P. larvae virulence on the larval level and that on the colony level are negatively correlated.American foulbrood (AFB) is among the economically most important honeybee diseases. The etiological agent of AFB is the gram-positive, spore-forming bacterium Paenibacillus larvae (9). The extremely tenacious spores are the infectious form of this organism. These spores drive disease transmission within colonies (11), as well as between colonies as soon as they end up in the honey stores of an infected colony (12).The species P. larvae can be subdivided into four different genotypes designated ERIC I to ERIC IV based on results from repetitive-element PCR (20) using enterobacterial repetitive intergenic consensus (ERIC) primers (9, 10), with P. larvae ERIC I and ERIC II being the two practically most important genotypes (1, 2, 9, 10, 13, 16). The four genotypes were shown previously to differ in phenotype, including virulence on the larval level (8, 9). While larvae infected with genotypes ERIC II to ERIC IV were killed within only 6 to 7 days, it took P. larvae ERIC I around 12 to 14 days to kill all infected individuals. Therefore, genotype ERIC I was considered to be less virulent and the other three genotypes were considered to be highly virulent (7-9) on the larval level.P. larvae is an obligately killing pathogen which must kill its host to be transmitted. The virulence of such an obligate killer is thought to be determined primarily by two factors, (i) the probability of infecting a host and (ii) the time to host death (6). The problem of ensuring a high enough probability of infecting the next host is solved for P. larvae by (i) the tenacious exospores, which remain infectious for over half a century (17) and, therefore, can wait for decades for the next host to pass by, and (ii) a high pathogen reproduction rate (23) and, thus, the production of an extremely high number of spores within each infected larva.For evaluating the second factor determining P. larvae virulence, the time to host death, it is important to consider the two levels of honeybee hosts, the level of the individual larva dying from AFB and the level of the colony succumbing to AFB.The virulence of P. larvae genotypes on the larval level has been analyzed recently (8, 9). We have now determined the colony-level virulence for the two most common and practically important (10, 16) genotypes of P. larvae, ERIC I and ERIC II, significantly differing in virulence on the larval level (8). We will discuss how the time to larval death relates to the time to colony death and how the hygie...