EMERGING PATHOGENS IN VERTEBRATES: BIOLOGY, GENOMICS AND INFECTIVITY OF BACTERIA ASCRIBED TO THE MIDICHLORIACEAE FAMILY

This PhD thesis is the outcome of a wider project focused on studying the biology, genomics and infectivity of bacteria ascribed to the Midichloriaceae family. Midichloriaceae are a group of bacteria extremely diversified in terms of the ecological and taxonomic diversity of their hosts. Members of the family Midichloriaceae can be considered as a wide bacterial group, still to be investigated under several aspects. In particular, the association of members of the family Midichloriaceae with parasites (such as ticks, fleas and parasitic amoebae) suggests that these bacteria could be potentially infectious to vertebrates (including humans) and even responsible for medical and veterinarian diseases. The researchers conducted in this PhD project have been focused on three main topics: i) possible involvement of a midichloriacea in a disease (red mark syndrome; RMS) affecting rainbow trout; ii) in-depth analysis of different aspects concerning the hard tick Ixodes ricinus and its intramitochondrial symbiont Midichloria mitochondrii (i.e. antibiotic treatment of the arthropod, salivary glands vs. ovaric tissue comparative proteomics, transmission of the symbiont to the vertebrate host); iii) presence of M. mitochondrii-related bacteria in different tick species. A brief description of each topic of the work done is here reported: i) RMS is a chronic skin disease of unknown etiology affecting farmed rainbow trout Oncorhynchus mykiss in Europe. It consists of single or multiple skin lesions usually localized on the trunk of fish approaching market size. Many authors suppose that a bacterial infection could be the cause of the disease. Some studies have reported a possible correlation between the presence of a Rickettsia-like organism (subsequently ascribed to the family Midichloriaceae) and RMS skin lesions. Our work concerning the possible involvement of a midichloriacea in RMS in rainbow trout resulted in the detection of bacteria of this family, besides in skin lesions, in various internal organs and in the bloodstream. No bacteria were found in unaffected fish and in healthy skin samples from affected individuals. Bacteria were detected also in fish ponds water were RMS-affected fish are farmed. These results give support to the hypothesis of the involvement of these bacteria in developing RMS. It is possible that some free-living eukaryotes could host or be the vector of this midichloriacea. ii) M. mitochondrii is the most prevalent symbiont of the hard tick I. ricinus, present in 100% of eggs and adult females of wild I. ricinus populations. This bacterium is intracellular and is the only known symbiont able to invade the mitochondria of the host cells. However, the role that M. mitochondrii plays in the host metabolism has yet to be elucidated. In addition, multiple lines of evidence indicate the possibility of the transmission of this bacterium to the vertebrate host during the tick blood meal. In order to investigate the role of M. mitochondrii in the biology of the tick host, we performed an antibiotic treatment on I. ricinus individuals, with the aim of reducing/eliminating the symbiont, and to potentially observe the dynamic of bacterial infection in the tick host. We microinjected engorged adult females of I. ricinus with tetracycline, and we allowed the resulting larvae to feed on gerbils treated with the same antibiotic. The amount of M. mitochondrii was evaluated at different stages of the experiment using molecular techniques. In addition we evaluated the presence/absence of the symbiont DNA in the blood of gerbils used for the larval feeding. The performed treatments did not allow to eliminate the symbiont population from the host tick, however it allowed to reduce the multiplication that occurs after the larval blood meal. These results open the way for future experiments, using different antibiotic molecules, different administration methods and antibiotic administration on subsequent tick stages, to fulfill the goal of eliminating M. mitochondrii from the host I. ricinus, a major step in our understanding of the impact of this bacterium on ticks. In order to investigate, from a proteomic point of view, the tick I. ricinus and its symbiont, the protein profile of the ovary tissue (OT) and of salivary glands (SG) of adult females of this tick species were generated. To compare the OT and SG profiles, 2-DE profiling followed by LC-MS/MS protein identification were performed. We detected 21 spots showing significant differences in the relative abundance between the OT and SG, ten of which showed 4- to 18-fold increase/decrease in density. This work allowed to establish a method to characterize the proteome of I. ricinus, and to detect multiple proteins that exhibit a differential expression profile in OT and SG. Additionally, it was possible to use an immunoproteomic approach to detect a protein from the symbiont. Finally, the method developed will pave the way for future studies on the proteomics of I. ricinus, with the goals of better understanding the biology of this vector and of its symbiont M. mitochondrii. The transmission of M. mitochondrii after I. ricinus bite has been evidenced through direct and indirect evidences in human and other vertebrates by different authors. However, the time of seroconversion against the antigens of this symbiont is still unknown, as well as the kinetic of the antibody response against M. mitochondrii. In this work we performed an experimental infestation of rabbits with wild I. ricinus ticks (harboring M. mitochondrii) and I. ricinus from a lab colony (free of the symbiont). We were able to show the first seroconversion of a vertebrate model against M. mitochondrii. The seroconversion occurs approximately around the first and the second week after tick infestation, with duration of at least one month after infestation. It has been observed that M. mitochondrii represent an actual pack of antigens that can persist in the vertebrate host for a prolonged time. M. mitochondrii DNA was also observed circulating in blood of infested rabbits. The flagellar protein FliD of M. mitochondrii can be considered an interesting marker for I. ricinus bite, at least concerning adult females bite. However, the antibody response against this protein suggests that a possible screening for this marker should be considered within a limited time after tick infestation. Additional markers both from M. mitochondrii and I. ricinus should be considered in view of developing a possible marker for I. ricinus bite detection. iii) The multiple screening of 17 tick species allowed the detection and quantification of bacteria of the family Midichloriaceae in seven of them, including the first report of a representative of this family in a soft tick species (Argasidae), Ornithodoros maritimus. Based on sequence identity and phylogenetic analysis we propose that all these bacterial symbionts of ticks could be members of the genus Midichloria. The performed screening highlights different prevalence levels and variable bacterial loads in different tick species including one, Ixodes aulacodi, where the bacterium is present in all examined individuals, like in I. ricinus. This result prompts us to hypothesize different roles of Midichloria bacteria in different tick species.