Development of TaqMan real-time PCR and droplet digital PCR protocols for the detection of Candidatus Midichloria mitochondrii and evaluation of exposure among wildlife.

Candidatus Midichloria mitochondrii (Ca. M. mitochondrii), an endosymbiont intracellular bacterium living in the mitochondria of several tick species, especially ixodid species, has been proposed as a potential marker for monitoring tick-bite exposure. Therefore, the present study aimed to develop two different diagnostic methods, TaqMan-based real-time PCR (rt-PCR) and Droplet Digital PCR (dd-PCR), targeting the 16 S rRNA gene and gyrB gene for the detection of Ca. M. mitochondrii in different wildlife species from several areas of southern Italy. Both techniques were validated using 10-fold serial dilutions of a sequenced positive control up to reach 10^-6 final dilution. Among wildlife field samples, both the techniques identified Ca. M. mitochondrii DNA, although dd-PCR showed higher sensitivity, being able to detect the target DNA in a higher dilution and in several spleen samples scored negative by rt-PCR. Noteworthy, these molecular methods revealed for the first time the presence of Ca. M. mitochondrii DNA in red foxes (Vulpes vulpes), Eurasian badgers (Meles meles), otters (Lutra lutra), porcupines (Hystrix cristata), European hares (Lepus europaeus), and alpacas (Vicugna pacos), suggesting a tick-bite exposure of these animals in the study area.