## 2 Materials and Methods
### Molecular Analysis
#### 2.3.2 TaqMan(r) Real-Time PCR Assay for the Detection of _P. larvae_ DNA in Sugar Samples  
To perform a highly specific and sensitive PCR, a new set of primers and a Taqman(r) probe (Table S1) were designed and used in the present study. They were designed using the Primer Express(r) software v.3.0 (Applied Biosystems, Foster City, CA, USA) according to the 16S rRNA gene sequences of _P. larvae_ strains of different ERIC genotypes available from GenBank (Table S2) in order to assess in silico inclusivity. The sequences were aligned by Clustal W, as implemented in BioEdit v.7.0.8.0 [46]. The specificity of the primers and probe was assessed in silico (exclusivity) via a BLAST search against 16S rRNA gene sequences of potential hive bacterial contaminants belonging to _Paenibacillus_, _Basillus_, and _Enterococcus_ genera available from GenBank (Table S3). The specificity of the primers and probe were additionally tested by real-time PCR against DNA extracted from 17 different bacteria species: _Paenibacillus alvei_, _P. thiaminolyticus_, _P. amylolyticus_, _P. azotofixanus_, and _P. aparius_; _Basillus thuringiensis_, _B. cereus_, _B. sphaericus_, _B. licheniformis_, _B. subtilis_, _B. punilus_, _B. megaterium_, _B. circulans_, _B. firmus_, and _B. polimixa_; _Brevibacillus latexospora_ as well as _Enterococcus faecalis_. All of the _P. larvae_ detection real-time PCR (real-time PCR) assays were performed with a QuantiFast Pathogen PCR + IC kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The amplification reactions were carried out in a final volume of 25 \(\upmu\)L containing 5 \(\upmu\)L of DNA extract, 1 X QuantiFast Pathogen PCR Master Mix with ROX, 350 nM of each forward primer, 700 nM of reverse primer, 200 nM of probe, 1 X Internal Control Assay (Qiagen, Hilden, Germany), and RNase-Free water. The thermal profile was composed of an initial denaturation step at 95 \({}^{\circ}\)C for 5 min, followed by 45 cycles of denaturation at 95 \({}^{\circ}\)C for 15 s and annealing/extension at 60 \({}^{\circ}\)C for 30 s, as recommended by the manufacturer's instructions of the Master Mix. A fluorescence signal was detected during the annealing/extension step at each cycle. All of the PCR assays were performed on a StepOnePlus(tm) Real-Time PCR System, Thermo Fisher Scientific (formerly Applied Biosystems(tm) (Waltham, MA, USA). At the end of each run all of the samples tested were evaluated for the presence of an amplification curve of the IC, and then evaluated for the presence/absence of a _P-larvae_-specific target curve. In the case of the absence of the IC curve, the samples were retested and diluted 1/10 in order to reduce the PCR inhibition factor. 3.3 Standard Curve and Quantitative TaqMan(r) Real-Time PCR Assay for the Absolute Quantification of _P. larvae_ Spores  
A spore suspension of the ERIC I P. _larvae_ ATCC 9545 strain in sterile ddH2O was prepared and counted in a Burker chamber at a light microscope (1000\(\times\)). Since the approximate spore counting method was based on the use of a Burker chamber, the count was performed by two independent readers, and the result was estimated as the average value of the two readings (data not shown). The spore suspension (5 \(\times\) 10\({}^{9}\) mL\({}^{-1}\)) was centrifuged at 9000\(\times\)\(g\) for 5 min, and the pellet was resuspended with Lysozyme solution, submitted to DNA extraction as described above, and the DNA was stored as stock at \(-\)20 \({}^{\circ}\)C. The number of copies of _P. larvae_ chromosomal DNA (5 \(\times\) 10\({}^{6}\) mL\({}^{-1}\)) in the stock was confirmed spectrophotometrically using the Synergy/HTX (BioTek Instruments, Inc., Winooski, VT, USA) by measuring the absorbance at wavelengths 260/280 nm, assuming the genome size of _P. larvae_ to be 4.29 \(\times\) 10\({}^{6}\) bases, according to the NCBI reference sequence NZ_CP019687.1, as reported in [47], corresponding to approximately 5 fg. The _P.