Data sets for evaluation of the virulence of Paenibacillus larvae DTK386 and DTK384, Paenibacillus sp. J27TS7, Paenibacillus azoreducens J34TS1, and Paenibacillus melissococcoides J46TS7 to honeybee larvae (Bioassay II).

Data sets (cumulative number of dead broods/number of larvae used [a-1, b-1, c-1, d-1, and e-1], survival rate [%] [a-2, b-2, c-2, d-2, and e-2], and the time taken to kill infected larvae [f-1 and f-2]) for evaluation of the virulence of Paenibacillus larvae DTK386 and DTK384, Paenibacillus sp. J27TS7, Paenibacillus azoreducens J34TS1, and Paenibacillus melissococcoides J46TS7 to honeybee larvae.The virulence was evaluated by feeding spores of these Paenibacillus strains to in vitro-reared Apis mellifera larvae for the first 24 h. P. larvae spores were prepared as previously described (https://doi.org/10.1292/jvms.19-0531). For preparation of the spores of the other bacteria, bacteria grown on BHI agar (J27TS7) and Columbia blood agar containing 5% sheep blood (CSA) (the other strains) were suspended in saline, and appropriately diluted bacterial suspensions were spread onto BHI agar (J27TS7) and CSA (the other strains) plates. The plates were incubated at 35°C for 11–16 days under aerobic conditions. After sporulation of more than 90% cells was confirmed by staining with malachite green, spores were collected from 10 or more agar plates, suspended in cold sterile H2O, and washed four times with cold sterile H2O by collecting spores via centrifugation (12,000 x g, 15 min, 4°C), discarding the supernatant, and suspending the spore pellet in 30 mL of cold sterile H2O. Washed spores were suspended in 10 mL of cold sterile H2O and stored at 4°C. One week or more later, the spores were washed several more times with 30 mL of cold sterile H2O to remove organic matter from the lysed vegetative cells. The re-washed spores were suspended in 5–20 mL of cold sterile H2O and stored at 4°C until use.Clinically healthy A. mellifera colonies maintained in the Research Institute for Animal Science in Biochemistry and Toxicology, Sagamihara, Japan, were used in this bioassay. From the colonies, less than 24-h-old larvae were grafted onto royal jelly in sterile Petri dishes using a grafting tool and randomly divided into test groups. Each larva was reared in grafting cells placed in a 48-well cell culture plate. The culture plates were kept in a desiccator (relative humidity, 95% ± 5%) and incubated at 34 ± 0.5ºC until day 6 post-grafting (pg). Larvae were infected by feeding them a diet containing spores on day 0 pg. Inoculum dose (i.e., concentration of spores in the diet) under each condition is shown in or on the top of each table. Larvae that received the inoculum on day 0 pg were transferred to wells with a bacteria-free diet 24 h later. Larvae in the non-infected control group were fed artificial diets containing the same components as the inoculum except the spores. On and after day 7 pg, larvae in the culture plates were incubated in a desiccator at 34 ± 0.5ºC (relative humidity, 80% ± 5%). On day 14 pg, each plate was transferred into an emergence box in a desiccator and incubated at 34 ± 0.5ºC (relative humidity, 80% ± 5%) until day 21 pg. In this bioassay, larval survival was monitored until day 21 pg.