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The M. plutonius strain used in this study was isolated from honeybee larvae and was deposited at the American Type Culture Collection (ATCC) (ATCC 35311) (Bailey and Collins, 1982).

The brain heart infusion (KHBHI) liquid medium (Table 1) and agar solid media (Table 2) were used for culturing M. plutonius in the laboratory. The anaerobic culture conditions were generated using BD BBL^TM GasPak^TM jar and BD GasPak^TM EZ Anaerobic Gas generating Pouch System with Indicator (Becton Dickinson, USA). For the liquid culture of M. plutonius, 50 mL of the KHBHI medium inoculated with bacteria was transferred to a 50-mL conical tube. Next, the tube and two gas generating packs were placed in the jar and the cap was quickly closed. The bacterial culture was incubated at 35℃ for 7 days. For the solid culture of M. plutonius, the bacteria were spread-plated on the KHBHI agar plate. Next, the plates were placed in the jar with the gas packs. The cultural conditions for solid culture were similar to those of the liquid culture.

The quantification of the M. plutonius number was performed under a light microscope (Primo Star binocular microscope, ZEISS, Jena, Germany) at 1000× magnification. The counting was performed in a microscopic field with an area of 0.05×0.05 mm². The counting was independently repeated 3 times and 20 areas were counted each time (total number of areas counted: 60 areas). The number of cells in one milliliter of suspension was calculated using the following formula:

Number of cell / mL
　=Total cells / (60×0.005 cm×0.005 cm×0.01 cm)

The DNA was isolated from M. plutonius using the DNeasy Blood & Tissue Kits (QIAGEN, Hilden, Germany), following the manufacturer’s instructions for isolating DNA from gram-positive bacteria. The bacterial cells were harvested by centrifuging the culture medium at 13,000×g for 1 min. The supernatant was discarded and the cell pellet was incubated with enzymatic lysis buffer comprising Tris-Cl (20 mM, pH 8.0), sodium EDTA (2 mM), Triton X-100 (1.2%), and lysozyme (20 mg/mL) at 37℃ for 30 min. Next, the cell lysate was incubated with proteinase K (25 μL) and lysis buffer (200 μL) at 56℃ for 30 min. The isolated DNA concentration was determined using a biophotometer (Eppendorf, Hamburg, Germany). To isolate the DNA from the infected honeybee samples, the larvae or pupae were ground in a 1.5-mL Eppendorf tube using a micropestle. Next, 100 μL of the lysis solution was transferred to a new tube and centrifuged at 13,000×g for 1 min. The supernatant was removed and the cell pellet was suspended in 180 μL of enzymatic lysis buffer. Next, the DNA was isolated following the same steps as those used for the isolation of DNA from pure bacterial culture.

The molecular detection and quantification of M. plutonius were performed in GENECHECKER ultra-rapid RT-PCR (UR RT-PCR; Genesystem Co., Ltd., Daejeon, Korea) using the premix 2X Rapi Mix (Genesystem Co.) containing SYBR green, a fluorescent dye for PCR detection. M. plutonius was detected using specific primers for the 16S ribosomal RNA gene, EF-DC-F1: 5′-AAG AGT AAC TGT TTT CCT CG-3′ and EF-DC-R1: 5′-TCC TCT TCT GCA CTC AAG TCT TC-3′ (Wang et al., 2016). The PCR conditions were as follows: 95℃ for 30 s, followed by 50 cycles 95℃ for 4 s, 53℃ for 4 s, and 72℃ for 4 s.

To calculate the initial DNA copy number, the recombinant DNA was serially diluted 10-fold (2.1×10⁸ to 2.1×10⁰ copies/μL). Next, 1 μL of each concentration was used for RT-PCR analysis. The standard linear regression representing the correlation between initial DNA copy and the corresponding threshold cycle (Ct) of amplification was established.

Total 1069 larvae of different ages of A. mellifera from three different honeycombs of the same hive were used for three independent infections. The first and second infection were done to evaluated the susceptibility of larvae of different ages (n=872) to the pathogen in an apiary, the second infection was done one week after the first infection. Other 197 larvae were used in the third infection one month after the first infection observe the response of A. mellifera colony to EFB pathogen and the health condition of the colony. The larval age was identified based on the shape and size of larvae in the cells according to the method of Human et al. (2013) (Fig. 1).

Fig. 1. 
Identification of larval age. The number “0” indicates the egg of honeybee, while the numbers 1~5 denote first to fifth larval instar stages (corresponding to the days 1~5 larvae), respectively. The number “6” indicates the day 6 larvae that are undergoing the process of capping. The white bar in the bottom right corner of each figure represents 1 cm.

For artificial infection of M. plutonius to the larvae, the bacterial cells were harvested by centrifuging the culture medium at 13,000×g for 30 s. The pellet was suspended in PBS solution and the bacterial concentration was determined by microscopic count. The bacterial suspension was serially diluted (1.335×10⁶ to 1.335×10¹ cells/μL) and 2 μL of different serially diluted bacterial samples (corresponding to the bacterial numbers of 2.67×10⁶ to 2.67×10¹) were added to each cell of the comb containing larvae of different ages using a multichannel pipette. The images of the combs containing the infected larvae were captured before infection and after the inoculation period. The artificial infection was performed within 30~45 min at room temperature. The combs with larvae were placed back into the hive and the larvae were allowed to be fed by the nurse bees. The infection was observed at day 4 and analyzed at day 7 post-infection.

In the first two infections, the larvae in the infected and surrounding areas were removed from the combs by nursing bees from day 4 to 7 post-infection. Only the cells with healthy larvae underwent capping and the larvae developed into the pupa stage. The capped cells were unsealed and the pupae were observed without any symptoms of EFB disease (Fig. 2). However, after the third infection adult bees in the infected colony were less active than those after the first two infections, and the ability of the adult bees to detect and remove the infected larvae decreased. Thus, only the larvae in the infected areas were removed on day 7 post-infection, whereas those in the surrounding areas were not removed. Although the symptom of EFB was not clearly identified in the hive, the PCR analysis of the healthy pupae and the larvae collected from the surrounding areas revealed the presence of M. plutonius (Fig. 3).

Fig. 2. 
Artificially infecting honeybee larvae with Melissococcus plutonius. The comb with the larvae used for the second infection is shown. The larvae of different ages in the four areas (indicated by numbers 1~4) were exposed to the pathogen at a concentration of 2.67×10⁶. The larvae in area 4 were exposed to different bacterial numbers (2.67×10⁶ to 2.67×10¹) (A). After one week, the infected larvae were eliminated by the nursing bee, which resulted in empty cells. The cells containing live larvae underwent capping (B). The capped pupae were unsealed to observe pupal health (C).

Fig. 3. 
Detection of Melissococcus plutonius from the infected larvae and pupae. The comb with the larvae used for the third infection is shown. The capped pupae exhibiting no symptoms of European foulbrood (EFB) disease in the infected area (the red line area) and the larvae in the surrounding areas were collected for the detection of M. plutonius by ultra-rapid real-time polymerase chain reaction (A). The amplification curves and melting curves revealed that M. plutonius was detected in the sample DNA (S). “P” is positive control containing 108 copies of M. plutonius recombinant DNA. “N” is the negative control without DNA template (B).

The eggs and days 1~6 larvae (n=552) in one comb that was infected twice were selected to calculate the effect of exposure to 2.67×10⁶ bacteria/cell on the survival rate. The number of surviving pupae in each age was counted on day 7 post-infection. The numbers of eggs, first instar, and second instar larvae removed after 7 days were 49, 4, 58, respectively. Of the 261 third instar larvae assessed, 4 larvae (1.53%) were alive on day 7 post-infection. The survival rate was high among the older larvae. The survival rates of 115 fourth instar larvae, 43 fifth larvae, 22 sixth instar larvae were evaluated, which revealed that 37.39, 46.51, and 59.09% of fourth, fifth, and sixth instar larvae developed into capped pupae, respectively.

The susceptibility of larvae to the disease was dependent on the larval age and not on the concentration of bacteria. The survival rate of larvae present in the areas treated with a low number of bacteria (2.67×10⁵ to 2.67×10¹) did not increase. The exposure of larvae to the pathogen at all tested concentrations markedly decreased the survival rates of first, second, and third instar larvae. The survival rates of fourth, fifth, and sixth instar larvae exposed to a high number of bacteria were higher than those of the younger larvae (Fig. 4).

Fig. 4. 
Infecting honeybee larvae with different pathogen numbers. Five areas (64 larvae in each area) used for the second infection are shown. The larvae were artificially infected with different numbers of M. plutonius (2.67×10⁵ to 2.67×10¹ bacteria/larva) (A). On day 7 post-infection, the infected larvae were eliminated from the comb and the surviving larvae developed into healthy pupae.

It is difficult to quantify the bacteria in the infected sample by microscopic counting due to the small size of bacteria and low purity of sample. In contrast, the qPCR analysis is highly rapid and sensitive for bacterial quantification. The limit of detection of recombinant DNA was 21 copies. The operation time of the UR RT-PCR system was 23 min 30 s for 50 cycles. The standard linear regression equation for the correlation between initial DNA copy and threshold cycle was y= -3.3584x+39.522; R²=0.9952 (where y and x were threshold cycle and log₁₀ (initial DNA copy), respectively).

The correlation between molecular and microscopic counts was also established using pure culture bacteria. The bacterial number was calculated by microscopic count in a bacterial culture with a concentration of 1.59×10⁸ cells/mL. The bacterial suspension was serially diluted 10-fold from 1.59×10⁸ to 1.59×10³ cells/mL. Next, 1 mL of each concentration was used for DNA isolation and calculation of DNA copy number by RT-PCR. The sample DNA copy numbers ranged from 4.74×10⁸±1.56×10⁸ to 4.22×10³±1.43×10³ copies (Table 3). The relationship between the results of two counting methods was represented by the linear equation y=1.0523x-0.113; R²=0.9868 (where x and y are log₁₀ number of the bacterial cell and DNA copy, respectively) (Fig. 5).

Fig. 5. 
Relationship between molecular count and microscopic count for the quantification of Melissococcus plutonius. Fluorescent curves show the amplification from DNA template that was isolated from 1.59×10⁸ to 1.59×10³ cells of M. plutonius, which are indicated by the numbers 8-3, respectively. “P” and “N” indicate positive control (recombinant DNA) and negative control (without DNA template), respectively (A). The relationship between the cell number and DNA copy number is represented by the linear regression (B).