Midichloria mitochondrii, an intracellular alphaproteobacteria considered as a symbiont of the tick Ixodes ricinus, found circulating in roe-deer (Capreolus capreolus) through molecular and serological evidences

The hard tick Ixodes ricinus, vector of pathogens important for both human and animal health (Parola and Raoult, 2001; Socolovschi et al., 2009) harbours Midichloria mitochondrii (order Rickettsiales; family Midichloriaceae), an endocytobiotic bacterium found in 100% of the females (Epis et al. 2008). Although its impact on the biology of its arthropod-host is still unknown, M. mitochondrii has first been detected in the reproductive apparatus of adult females where it is the most abundant and, more recently, also in the salivary glands of the parasite, suggesting the hypothesis of its transmission to the vertebrate host during the tick bite (Mariconti et al., 2012). To validate this hypothesis, the presence of M. mitochondrii DNA in the vertebrate hosts (dogs, sheep, horses and humans) was detected through the amplification of the 16S rDNA gene (Mariconti et al., 2012; Bazzocchi et al. 2013) and serological analysis on sera collected from humans and dogs exposed to the tick bite (Mariconti et al. 2012). This experimental work aims to investigate the circulation of M. mitochondrii in roe deer (C. capreolus), which is the host of choice especially for the adult stage of I. ricinus in Europe and is therefore considered an efficient animal-sentinel for possible etiologic agents transmitted by this hard tick species. Briefly, we evaluate: 1) the presence of circulating M. mitochondrii DNA in blood of seven roe deer, different in age and sex, from a defined and controlled area (South-East Toulouse, France); 2) the immunological response of the roe deer individuals against a specific recombinant flagellar protein of M. mitochondrii (rFliD). Molecular analysis were conducted using specific qualitative PCRs for 12S rDNA of C. capreolus (Fajardo et al. (2008) and 16S rDNA of M. mitochondrii ( Epis et al. 2008) after DNA extraction from blood samples. Immunological analysis were conducted using the recombinant protein rFLID as antigen in ELISA and Western blot testsAfter molecular analysis, only three of the seven analyzed blood samples were positive to M. mitochondrii DNA. On the base of the already set cut-off (designed on negative sera), six out of seven roe deer individuals resulted positive in ELISA. Western blot assay instead, showed the presence of a band, at the expected molecular weight, in all the examined samples, except for the negative control. In conclusion, M. mitochondrii was detected in roe deer individuals through molecular and serological analyses, proving the circulation of M. mitochondrii in C. capreolus parasitized by I. ricinus. As already demonstrated in other studies, roe deer is a good subject to study the spread of tick-borne pathogens (or potentially pathogen). Further studies will allow the understanding of biological interactions of M. mitochondrii, the possible replication of this bacterium inside the host and its role in possible pathological alterations after its transmission