## 3 Results and Discussion
Previous in vivo studies could not ascertain such a contamination route, since the experimental design allowed the mites to be infected following the bloodmeas or by contact with feces or other excretes [15]. In fact, both the transcuitular absorption of surface pathogens and their entry through the stigmata, followed by an internal infection, have been suggested [28]. In vitro devices offer the possibility to confine the mites in a separate environment, focusing on blood as the only infection source. Such an approach confirmed the oral entry of \(S\). Gallinarum, despite it not being able to exclude the other route. A further, not negligible advantage is the possibility to avoid the use of animal models for investigating the transmission of pathogens mediated by hematophagous mites, such as \(D\). _gallinae_. The topic raises great interest, insomuch that the vectorial role has been widely investigated during the decades through the adoption of in vitro and in vivo approaches [7]. In vivo studies are usually considered more reliable, but they need to deal with live animals, with all the related ethical concerns. On the other side, most of the in vitro alternatives showed several drawbacks. Among the first methods to be described, one-day-old chick skin has proven to be effective, allowing the assessment of the vectorial role of the PRM for \(S\). Enteritidis [14]. In any case, this option does not solve the problem of using live animals, as chicks must be sacrificed to obtain their skin, and the technique was time-consuming. On the other hand, the synthetic membranes tested (e.g., Nescofilim, Parafilm, or rayon) did not ensure the same results of chick skin in terms of feeding rates [28]. Goldbeater's \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Life Stage** & **Specimens per** & **Samples Tested** & **qPCR-Positive** & **qPCR-Positive** \\  & **Sample (\(n\))** & **(\(n\))** & **Samples (\(n\))** & **Samples (\%)** \\ \hline Adult & 5 & 19 & 17 & 89 \\ Egg & 1 & 41 & 2 & 5 \\ Egg & 4 & 23 & 4 & 17 \\ Larva & 5 & 11 & 1 & 9 \\ Protonymph & 5 & 7 & 3 & 43 \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Detection of _Salmonella enterica_ ser. Gallinarum in the tested _Dermanysuss gallinae_ at different life stages. skin (Baudruche membrane) has been proposed as a valid alternative to chick skin, offering a feeding rate of 50% with goose blood as food source [21]. Although made by bovine intestine, this membrane is commercially available as preservation equipment. In this case, the in vitro feeding system allowed us to successfully confirm that _D. gallinae_ can acquire \(S\). Gallinarum from infected blood, and to strongly suggest the transovarial and trans-stadial transmission of the pathogen, as previously demonstrated for \(S\). Enteritidis [29]. In this case, the sensitivity of pathogen detection methods might represent an experimental weakness. Thus, the detection technique proposed herein ensured to successfully test PRM aliquots composed of few specimens (4-5) and even single eggs. The first studies about the vectorial role of _D. gallinae_ were mostly performed detecting pathogens through cultural [19; 28] or immunological methods [29]. The application of molecular techniques (i.e., PRC and qPCR) allowed to reach more refined results about this topic, especially in terms of quantification of pathogens within the mites [8; 15; 20]. However, these studies are usually made from pools each of 50-100 individuals, hence gathering only estimated information about the pathogen load per mite. The main issue relies on obtaining good quality DNA suspensions, due to the small size of the PRM and the presence of amplification inhibitors [30]. Several extraction methods have been proposed, such as cethyl trimethyl ammonium bromide buffer, a Chelex resin, a filter-based technology, and commercial kits, with the latter as the most effective and widely used [29; 30]. Here, we obtained good quality results adopting two different DNA extraction methods, depending on the PRM life stage.