*4.2. Extraction of Total Nucleic Acids*

All the SHBs were washed with 95% ethanol to remove possible external microbial contaminants. The ethanol was then allowed to evaporate at room temperature.

A TissueLyser II (Qiagen, Hilden, Germany) was used for 3 min at 25 Hz to crush all SHB samples in separate 2 mL Eppendorf tubes filled to the mark with RNase-free water. The resulting suspensions were then split into two equal aliquots from which nucleic acids were extracted (one for DNA and one for RNA).

DNA and total RNA were extracted with DNeasy Blood & Tissue Kit (Qiagen) and RNeasy Mini Kit (Qiagen) as previously described [20,67]. All samples were eluted in 30 μL DNAase-RNase-free water.

DNA and RNA extracts were stored at −80 ◦C until analysis. High pure sterile DNAand RNA-free water was used as a negative control in all analytical steps.

#### *4.3. PCR Assays to Detect Bacteria and Protozoa DNA*

The extracted DNA was analyzed by real-time PCR to detect bacteria and trypanosomatids. The primers that were used are reported in Table 2.

For each target gene, a total reaction volume of 15 μL was prepared as previously described [81] using 2x QuantiTect Probe PCR Master Mix (Qiagen), forward and reverse primers (2 μM), forward and reverse probes (500 nM), and 3 μL DNA extract. The real-time PCR assay was performed on a Rotorgene Corbett 6000 (Corbett Research, Sydney, Australia) following the protocols for either gene sequence [54,68]. DNA extracted previously from positive honey bees was used as the positive control for each investigated bacterial and protozoan species.


