Molecular and serological evidences of circulating Midichloria mitochondrii in roe deer (Capreolus capreolus) after Ixodes ricinus bite

Midichloria mitochondrii (order Rickettsiales; family Midichloriaceae) is an intracellular bacterium found in the reproductive apparatus and in the salivary glands of 100% of adult females of the hard tick Ixodes ricinus, vector of pathogens important for both human and animal health. Furthermore, direct (DNA amplification) and indirect (immunological analysis) evidences are published on the presence of M. mitochondrii in blood and serum of different mammalian hosts, including humans, suggesting a transmission during the tick bite. This experimental work aims to investigate the circulation of M. mitochondrii in roe deer (C. capreolus), which is the host of choice for adult and nymph stages of I. ricinus. Briefly, we evaluated: 1) the presence of circulating M. mitochondrii DNA in blood of seven roe deer from the INRA Gardouch experimental station (Haute Garonne; France); 2) the immunological response of the same roe deer individuals against a recombinant flagellar protein of M. mitochondrii (rFliD). Blood and sera were collected, from the same animals, during the spring of 2014 and 2015. DNA was extracted from blood samples and molecular analysis were conducted using a specific qualitative PCR for 16S rDNA of M. mitochondrii. Instead, immunological analysis were conducted using the recombinant protein rFLID as antigen in ELISA assay. After molecular analysis, positivity to M. mitochondrii DNA was found in some blood samples collected both in 2014 and 2015 springs. Furthermore, positivity in ELISA test was found in six out of seven roe deer sera collected in 2014, and in all samples collected in 2015. The specificity of these results was also confirmed by a Western blot analysis. The obtained results demonstrate the circulation of M. mitochondrii in the blood of C. capreolus parasitized by I. ricinus, confirming once again that this mammalian host is a good subject to study the spread of tick-borne pathogens.