Diversity of Melissococcus plutonius from Honeybee Larvae in Japan and Experimental Reproduction of European Foulbrood with Cultured Atypical Isolates

European foulbrood (EFB) is an important infectious disease of honeybee larvae, but its pathogenic mechanisms are still poorly understood. The causative agent, Melissococcus plutonius, is a fastidious organism, and microaerophilic to anaerobic conditions and the addition of potassium phosphate to culture media are required for growth. Although M. plutonius is believed to be remarkably homologous, in addition to M. plutonius isolates with typical cultural characteristics, M. plutonius-like organisms, with characteristics seemingly different from those of typical M. plutonius, have often been isolated from diseased larvae with clinical signs of EFB in Japan. Cultural and biochemical characterization of 14 M. plutonius and 19 M. plutonius-like strain/isolates revealed that, unlike typical M. plutonius strain/isolates, M. plutonius-like isolates were not fastidious, and the addition of potassium phosphate was not required for normal growth. Moreover, only M. plutonius-like isolates, but not typical M. plutonius strain/isolates, grew anaerobically on sodium phosphate-supplemented medium and aerobically on some potassium salt-supplemented media, were positive for β-glucosidase activity, hydrolyzed esculin, and produced acid from L-arabinose, D-cellobiose, and salicin. Despite the phenotypic differences, 16S rRNA gene sequence analysis and DNA-DNA hybridization demonstrated that M. plutonius-like organisms were taxonomically identical to M. plutonius. However, by pulsed-field gel electrophoresis analysis, these typical and atypical (M. plutonius-like) isolates were separately grouped into two genetically distinct clusters. Although M. plutonius is known to lose virulence quickly when cultured artificially, experimental infection of representative isolates showed that atypical M. plutonius maintained the ability to cause EFB in honeybee larvae even after cultured in vitro in laboratory media. Because the rapid decrease of virulence in cultured M. plutonius was a major impediment to elucidation of the pathogenesis of EFB, atypical M. plutonius discovered in this study will be a breakthrough in EFB research

Results of DNA-DNA hybridization.

a<p>Because % similarities were shown as the degree of DNA-DNA reassociation calculated based on OD₄₀₅ values obtained as the result of enzyme reaction, they can be greater than 100%.</p>b<p><i>M. plutonius</i>-like isolates.</p>c<p><i>M. plutonius</i> isolates.</p>d<p><i>E. faecalis</i> type strain.</p

Formulas of culture media used in this study.

<p>Unit: g/L.</p><p>Medium 1 to 6 and carbohydrate test media were autoclaved at 115°C for 10 min. Other media were autoclaved at 121°C for 15 min.</p>a<p>The pH was adjusted to 6.6 with KOH.</p>b<p>The pH was adjusted to 6.6 with the solution, in which the mole ratio of KOH/NaOH was 1∶1.</p>c<p>The pH was adjusted to 6.6 with HCl.</p>d<p>The pH was adjusted to 6.6 with NaOH.</p>e<p>Potassium and sodium salts were added to the media to final concentrations of 0.033 M (Medium 4), 0.1 M (Medium 1, 3, 5 and 6) or 0.15 M (KSBHI and KBHI).</p>f<p>D-cellobiose, lactose, D-raffinose, or D-xylose</p>g<p>After the base medium was autoclaved, the carbohydrate was added aseptically.</p

Dendrogram of SmaI-digested PFGE profiles of typical and atypical <i>M. plutonius</i> strain/isolates.

<p>Phenotypically distinct strain/isolates were also grouped separately into two distinct genetic clusters.</p

Culture characteristics of <i>M. plutonius</i> and <i>M. plutonius</i>-like strain/isolates used in this study.

a<p>The growth of ATCC 35311 cultured on KSBHI agar under anaerobic conditions was scored as +. Compared to this growth, more vigorous and weaker growth was scored as +^v and +^w, respectively. No growth or only trace levels of growth was scored as −.</p

Biochemical characteristics of <i>M. plutonius</i> and <i>M. plutonius</i>-like strain/isolates used in this study.

a<p>Repeated tests showed slightly different results in ATCC 35311.</p>b<p>Mannitol, sucrose, maltose, D-xylose, glycerin, D-melezitose, D-raffinose, D-sorbitol, L-rhamnose, and D-trehalose.</p

Isolation and characterization of patient-derived, toxic, high mass amyloid beta-protein (Abeta) assembly from Alzheimer disease brains.

Amyloid beta-protein (Abeta) assemblies are thought to play primary roles in Alzheimer disease (AD). They are considered to acquire surface tertiary structures, not present in physiologic monomers, that are responsible for exerting toxicity, probably through abnormal interactions with their target(s). Therefore, Abeta assemblies having distinct surface tertiary structures should cause neurotoxicity through distinct mechanisms. Aiming to clarify the molecular basis of neuronal loss, which is a central phenotype in neurodegenerative diseases such as AD, we report here the selective immunoisolation of neurotoxic 10-15-nm spherical Abeta assemblies termed native amylospheroids (native ASPDs) from AD and dementia with Lewy bodies brains, using ASPD tertiary structure-dependent antibodies. In AD patients, the amount of native ASPDs was correlated with the pathologic severity of disease. Native ASPDs are anti-pan oligomer A11 antibody-negative, high mass (&gt;100 kDa) assemblies that induce degeneration particularly of mature neurons, including those of human origin, in vitro. Importantly, their immunospecificity strongly suggests that native ASPDs have a distinct surface tertiary structure from other reported assemblies such as dimers, Abeta-derived diffusible ligands, and A11-positive assemblies. Only ASPD tertiary structure-dependent antibodies could block ASPD-induced neurodegeneration. ASPDs bind presynaptic target(s) on mature neurons and have a mode of toxicity different from those of other assemblies, which have been reported to exert their toxicity through binding postsynaptic targets and probably perturbing glutamatergic synaptic transmission. Thus, our findings indicate that native ASPDs with a distinct toxic surface induce neuronal loss through a different mechanism from other Abeta assemblies