Characterization of some enzymatic properties of recombinant α-glucosidase III from the Thai honeybee, Apis cerana indica Fabricus

Recombinant α-glucosidase III (rHBGase III) from Apis cerana indica Fabricus (rAciHBGase III) was expressed in the yeast Pichia pastoris GS115, enriched and characterized. The full length cDNA of AciHbgase III (ƒî1.8 kb) was amplified by RT-PCR, cloned into the pPICZαA expression vector and used to transform P. pastoris GS115. The maximum secreted expression level of rAciHBGase III [as an N terminal (His)6 tagged chimera] was found 144 h after induction by 1% (v/v) methanol. Enrichment of the enzyme using histrap affinity purification revealed a single active glucosidase band with a molecular mass of ~68 kDa. The optimal pH and temperature for glucosidase activity of the enriched rAciHBGase III were pH 5.0 and 37¢XC, respectively, whilst the enzyme showed a good pH stability between pH 5.0 to 7.5, but not below pH 5.0, and a poor thermotolerance with < 10% and 0% residual activity at 40 and >50¢XC, respectively. The rAciHBGase showed a relatively high substrate specificity for maltose (Km of 4.5 mM) and p-nitrophenyl α-D-glucoside (Km of 4.4 mM) compared to other reported HBGase enzymes.Key words: α-Glucosidase, Apis cerana indica, expression, kinetics, recombinant enzyme

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

Differential susceptibility to the tracheal mite Acarapis woodi between Apis cerana and Apis mellifera

International audienceAbstractIn Japanese honey bees Apis cerana japonica, infestations of the tracheal mite Acarapis woodi have spread rapidly over the mainland of Japan, causing damage and the collapse of colonies. Meanwhile, infestations by mites in Apis mellifera have hardly been observed in Japan. In this study, we assessed and compared the susceptibility of the two species, A. cerana and A. mellifera, using an inoculation assay. We found that migrating female mites entered the tracheae of more newly emerged bees in both species but more frequently in A. cerana than in A. mellifera. Hence, the higher susceptibility in A. cerana is proposed as a factor causing the explosive epidemic of tracheal mites in only A. cerana in Japan. Moreover, we compared grooming behaviors between the two bee species using an observation assay as a preliminary experiment, although the bees were not exposed to the presence of tracheal mites. From these observations, the frequency of autogrooming (self-grooming) on the thorax in A. cerana was lower than that in A. mellifera. The difference in susceptibility to the mite between these two species may be due to the difference in grooming behavior frequency

A case of disseminated M. bovis bacillus Calmette-Guérin (BCG) disease after one month of BCG bladder infusion therapy and analysis of 77 cases of suspected BCG infection in Japan, 2017–2022

Bacillus Calmette-Guerin (BCG) intravesical injections are used as adjuvant therapy for superficial bladder cancer. We report a case of a 78-year-old man who developed disseminated M. bovis BCG disease mimicking miliary tuberculosis early after BCG intravesical infusion. He started coughing after receiving three rounds of BCG for superficial bladder tumors, following transurethral resection of the tumors, approximately one month after initiation. Computerized tomography (CT) images showed diffuse nodular shadows in the bilateral lung fields with a random pattern. Consequently, disseminated BCG disease was diagnosed. Treatment with isoniazid, rifampicin, and ethambutol was initiated. Nine months after initiating treatment, CT showed the disappearance of the miliary shadows. We also discussed 77 cases of suspected BCG infection and the requests for Mycobacterium bovis BCG identification at our institution from 2017 to October 2022. Of these, 76 cases were M. bovis BCG, and 1 case was M. tuberculosis. Since M. tuberculosis can be identified in some patients with suspected BCG infection, it is crucial to distinguish between the two based on pathogenicity