DOI: 10.6084/m9.figshare.26941720
title: Nakamura, K., Okamoto, M., Mada, T., Harada, M., Okumura, K., & Takamatsu, D. Data sets for comparison of the virulence of <i>Paenibacillus</i> sp. J27TS7 and <i>Paenibacillus melissococcoides</i> strains to honeybee larvae (Bioassay III).

description: Data sets (cumulative number of dead broods/number of larvae used [a-1, b-1, c-1, and d-1], survival rate [%] [a-2, b-2, c-2, and d-2], larval weight [e], and bacterial load [CFU/mg larva] [f]) for evaluation of the virulence of <i>Paenibacillus</i> sp. J27TS7 and <i>Paenibacillus</i> <i>melissococcoides </i>J1TS6, J6TS7, J10TS2, J11TS3, J22TS4, and J46TS7 to honeybee larvae.The virulence was evaluated by feeding spores of these <i>Paenibacillus</i> strains to <i>in vitro</i>-reared<i> Apis mellifera</i> larvae. For preparation of the spores, bacteria grown on BHI agar (J27TS7) and Columbia<b> </b>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), MYPGP agar (J6TS7), and CSA (the other strains) plates. The plates were incubated at 35°C for 11–16 days under air plus 5% CO<sub>2</sub> (J6TS7) and aerobic (the other strains) 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 H<sub>2</sub>O, and washed four times with cold sterile H<sub>2</sub>O 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 H<sub>2</sub>O. Washed spores were suspended in 10 mL of cold sterile H<sub>2</sub>O and stored at 4°C. One week or more later, the spores were washed several more times with 30 mL of cold sterile H<sub>2</sub>O to remove organic matter from the lysed vegetative cells. The re-washed spores were suspended in 5–20 mL of cold sterile H<sub>2</sub>O and stored at 4°C until use.Clinically healthy<i> A. mellifera</i> 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 or day 2 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, and larvae that received the inoculum on day 2 pg were reared in the same wells thereafter. Larvae in the non-infected control group were fed artificial diets containing the same components as the inocula 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, two groups of larvae were tested for each strain. One was used to assess survival, and the other was used to measure larval weight and bacterial load and/or to analyse histopathologically. After measuring the weights, larvae were stored in a deep freezer at -80°C (for measuring bacterial load) or rinsed for several seconds with sterilised distilled water and fixed in 10% phosphate-buffered formalin (for histopathological analysis). Bacterial loads in the infected larvae were calculated by plating serial dilutions of larval homogenates onto CSA plates and counting colonies on the plates after incubation of the plates at 35ºC for seven days under aerobic conditions. Histopathological analysis results are not included in the data sets. In this bioassay, larval survival was monitored until day 21 pg.