UNIVERSITÀ DEGLI STUDI DI MILANO Emerging Pathogens In
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UNIVERSITÀ DEGLI STUDI DI MILANO PhD Course in Veterinary and Animal Science (Class XXIX) Department of Veterinary Medicine (DIMEVET) Emerging pathogens in vertebrates: biology, genomics and infectivity of bacteria ascribed to the Midichloriaceae family Alessandra CAFISO No. R10530 Tutor: Prof. Chiara BAZZOCCHI Coordinator: Prof. Fulvio GANDOLFI Academic Year 2015-2016 Index List of abbreviations ................................................................................................................. 4 Abstract ..................................................................................................................................... 5 Riassunto ................................................................................................................................... 8 1 General introduction ....................................................................................................... 11 1.1 The bacterial family Midichloriaceae ..............................................................................11 1.2 Midichloriaceae in ticks and transmission to vertebrate hosts ....................................... 14 1.2.1 Ticks and transmission of bacteria: a brief general description .................................... 14 1.2.2 Midichloria mitochondrii in Ixodes ricinus ................................................................... 18 1.2.3 Midichloriaceae in other tick species ............................................................................ 20 1.2.4 Transmission of Midichloriaceae from ticks to vertebrate hosts .................................. 22 1.3 Involvement of a bacterium of the family Midichloriaceae in rainbow trout Oncorhynchus mykiss affected by red mark syndrome .............................................................. 24 2 Purpose of the PhD project ............................................................................................ 29 3 Performed researches ...................................................................................................... 31 3.1 Molecular evidence for a bacterium of the family Midichloriaceae (order Rickettsiales) in skin and organs of the rainbow trout Oncorhynchus mykiss (Walbaum) affected by red mark syndrome .................................................................................................................................... 31 3.1.1 AIM OF THE STUDY .................................................................................................. 31 3.1.2 MATERIALS AND METHODS ................................................................................... 31 3.1.3 RESULTS AND DISCUSSION .................................................................................... 32 3.2 Antibiotic treatment of the hard tick Ixodes ricinus: influence on Midichloria mitochondrii following blood meal ............................................................................................ 37 3.2.1 AIM OF THE STUDY .................................................................................................. 37 3.2.2 MATERIALS AND METHODS ................................................................................... 38 3.2.3 RESULTS ...................................................................................................................... 41 3.2.4 DISCUSSION ............................................................................................................... 45 3.3 Molecular screening for Midichloria in hard and soft ticks reveals variable prevalence levels and bacterial loads in different tick species ...................................................................... 48 3.3.1 AIM OF THE STUDY .................................................................................................. 48 3.3.2 MATERIALS AND METHODS ................................................................................... 48 3.3.3 RESULTS ...................................................................................................................... 52 3.3.4 DISCUSSION ............................................................................................................... 56 3.3.5 SUPPORTING INFORMATION .................................................................................. 60 2 3.4 Ixodes ricinus and its endosymbiont Midichloria mitochondrii: a comparative proteomic analysis of salivary glands and ovaries ....................................................................................... 62 3.4.1 AIM OF THE STUDY .................................................................................................. 62 3.4.2 MATERIALS AND METHODS ................................................................................... 62 3.4.3 RESULTS ...................................................................................................................... 67 3.4.4 DISCUSSION ............................................................................................................... 76 3.4.5 SUPPORTING INFORMATION .................................................................................. 80 3.5 Study of the seroconversion against antigens of the endosymbiont Midichloria mitochondrii in rabbits parasitized by Ixodes ricinus ................................................................. 89 3.5.1 AIM OF THE STUDY .................................................................................................. 89 3.5.2 MATERIALS AND METHODS ................................................................................... 90 3.5.3 RESULTS ...................................................................................................................... 96 3.5.4 DISCUSSION AND CONCLUSIONS ....................................................................... 102 3.6 Blood circulation and presence in the subcutaneous muscle layer of a Midichloria-like organism (MLO) associated with red mark syndrome in the rainbow trout Oncorhynchus mykiss (Walbaum) ................................................................................................................................ 106 3.6.1 AIM OF THE STUDY ................................................................................................ 106 3.6.2 MATERIALS AND METHODS ................................................................................. 106 3.6.3 RESULTS .................................................................................................................... 109 3.6.4 DISCUSSION .............................................................................................................. 112 4 Conclusion of the PhD project and future perspectives ............................................ 115 5 References ...................................................................................................................... 117 6 List of publications ........................................................................................................ 129 3 List of abbreviations 2DE, 2-DE: two dimensional electrophoresis cal: calreticuline ELISA: enzyme-linked immunosorbent assay EM: electronic microscopy FISH: fluorescence in situ hybridization FliD: the flagellar cap protein (100 kDa) GAPDH: glyceraldehyde 3-phosphate dehydrogenase gyrB: gyrase B igf1: insulin growth factor 1 MLO, MALO: Midichloria-like organism PCR: polymerase chain reaction qPCR: quantitative real time PCR rFliD: recombinant form of the flagellar cap protein (38 kDa) RLO: Rickettsia-like organism RMS: red mark syndrome SD: strawberry disease SG: salivary gland OT: ovaric tissue 4 Abstract 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