## 2 Materials and Methods
### Molecular Detection of Salmonella Gallinarum in Mites  
DNA was extracted from \(D\). _gallinae_ specimens with different methods, depending on the life stage. Adults, larvae, and protonyms were grouped in pools, each of 5 specimens of the same stage. The DNA was extracted with the ZymoBIOMICS DNA Miniprep Kit (Zymo Research, Irvine, CA, USA), according to the manufacturer's instructions, in a final volume of 50 mL. The obtained DNA solution was divided into two aliquots, each of 25 mL, centrifuged at 16,000\(\times\)_g_ for 10 m at 4 \({}^{\circ}\)C, and then dried at 65 \({}^{\circ}\)C. The resulting pellet was resuspended in 12 or 6 mL TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0).  
The eggs were placed singularly or in pools of four specimens in a sterile tube with 12 or 6 mL of sterile water and squashed with a sterile wooden toothpick. Then, the solution was heated at 90 \({}^{\circ}\)C for 10 m to cause the egg rupture releasing DNA.  
For all the PRM stages, the final volume of DNA solution was calculated in order to be entirely used for the following investigation, reducing at the minimum the material loss.  
The detection of S. Gallinarum in mites was performed by a multiplex qPCR with primers and probe previously described [20] and, as an internal control, primers and probe for \(D\). _gallinae_ designed using Primer3 software [22] on the basis of distinctive regions of \(D\). _gallinae_ (GenBank accession no. FN599090.1). Both primers and probe were checked for self-complementarity, hetero-complementarity, and melting temperature according to MIQE guidelines [23]. All primers and probes are listed in Table 1.  
The multiplex qPCR was carried out on CFX Connect Real-Time PCR Detection System (Bio-Rad, Milan, Italy) in 96-well optical plates (Bio-Rad). Amplification was performed in a final volume of 15 mL, comprising SsoAdvanced Universal Probes Supermix (Bio-Rad), 20 nM of each primer-probe solution (Bio-Rad), and 3 mL of template. Non-template controls (NTC) were included in each run, adding an equal amount of sterile water instead of DNA solution. The amplification conditions were as follows: 95 \({}^{\circ}\)C for 3 m; 40 cycles  
\begin{table}
\begin{tabular}{l l l} \hline \hline
**Oligomer Name** & **Nucleotide Sequence, 5\({}^{\prime}\) to 3\({}^{\prime}\)** & **Length (bp)** & **Reference** \\ \hline RTSGf & CCGATATGAGGATTAC & 144 & \\ RTSGr & AGGTCGTAATGACAAA & [20] \\ RTSGp & FAM-ACATCGTAATTCATGCACTACCCAT & \\ RTELFaF & GTTCGAAGCTGATCTC & 183 & \\ RTELFaR & TAGCCGATTCTTGTGATG & This study \\ RTELFaP & HEX-ACAGCGACACCTCTCTCTTGAT & — \\ \hline \hline \end{tabular}
\end{table}
Table 1: Primers and probes used for the amplification of _Salmonella_ Gallinarum and _Dermurysss gallinae._of 95 \({}^{\circ}\)C for 10 s, 55 \({}^{\circ}\)C for 15 s; 60 \({}^{\circ}\)C for 15 s. Fluorescence from both FAM and HEX channels was acquired during each extension step. Cycle threshold (CT) and baseline were calculated automatically by the CFX Maestro Software v.5.2 (Bio-Rad). The samples were considered positive when the CT was lower than 35. Each DNA solution and the NTCs were amplified in quadruplicate or duplicate in the same plate.