Can protist ciliates act as a natural reservoir for bacteria potentially pathogenic for Metazoa? Trans-infection experiments of Rickettsiales endosymbionts from the ciliates Euplotes and Paramecium to the planarian Dugesia

Most of the microorganisms of genera responsible for vector-borne diseases (VBD) has hematophagous arthropods as vector/reservoir. Recently, many new species of such microorganisms were found in various terrestrial and aquatic eukaryotic hosts: numerous new bacterial species belonging/related to the genus Rickettsia(Alphaproteobacteria, Rickettsiales; with obligate intracellular lifestyle) were discovered in protist ciliates. Although their pathogenicity for either humans or animals is still under study, these bacteria could actually act as etiological agents of possible VBD in aquatic environment. Indeed, an increasing number of massive fish death was recorded in intensive aquaculture facilities during the lasts years due to epidemics caused by Rickettsia-like bacteria with unknown natural vector. As ciliates could vector pathogenic organisms possibly responsible for zoonosis, this study was meant to verify the transmission of the Rickettsiales endosymbionts hosted by two species of ciliates to a metazoan model, the planarian Dugesia japonica. The ciliates were Euplotes woodruffi and Paramecium multimicronucleatum; the first hosts in the cytoplasm two differentRickettsiales endosymbionts, Candidatus (Ca) Megaira polyxenophila and Ca. Bandiella woodruffiiâ, and the betaproteobacterium Polynucleobacter necessarius; the second hosts in the macronucleus a subspecies of the Rickettsiales endosymbiont Ca. Trichorickettsia mobilis. Ciliate monoclonal mass cultures were set up for trans-infection experiments via os: 1. Washed, concentrated ciliates were homogenized, pelleted, and added to regular food for planarians. 2. Antibiotic-treated planarians were fed on ciliate-enriched food or regular food (control), washed, and let digest for 1, 2, 3, and 7 days. 3. A comparative multidisciplinary investigation through DNA extraction-PCR procedure, and TEM observation was performed. DNA of endosymbionts was recovered up to 7 days after feeding in treated planarians, and we could identified bacteria at TEM in treated planarians intestine also outside digestive vacuoles, indicating that they somehow are able to avoid animal digestion