DEVELOPMENT OF A SEROLOGICAL TEST FOR IXODES RICINUS BITE IN VERTEBRATES

The research line of my PhD project was focused on parasitism supported by Ixodes ricinus tick, the most common tick species occurring in Europe infecting humans and animals. The importance of this tick species is due to its wide area of distribution (i.e. Europe and Northern Africa), to its low host specificity and to its central role in the transmission of multiple infectious agents (e.g. the causative agent of Lyme borreliosis, rickettsial diseases, tick-borne encephalitis (TBE) virus) to both humans and animals. In addition to these pathogens, I. ricinus hosts an intracellular bacterium named Midichloria mitochondrii to recall its unique intra-mitochondrial lifestyle. Indeed, this bacterium presents the peculiar capacity to invade and multiply inside mitochondria, where it has been found between the inner and outer membranes and consuming the mitochondrial matrix. The presence of M. mitochondrii has been detected in female reproductive tract, in salivary glands and rostrum of adult females of I. ricinus, where is present with a prevalence of 100%, while the prevalence in males is around 44%. A vertical transmission of the bacterium has been reported, as indicated by PCR on eggs and newly emerged larvae, together with its horizontal transmission to vertebrate host during I. ricinus blood meal, as demonstrated by molecular and serological evidences in different host species (including humans). Even though the transmission of M. mitochondrii into the vertebrate hosts during the I. ricinus bite is currently established, it is still unknown whether immature stages (larvae and nymphs) of this tick species transmit the symbiont during the blood meal. Additionally, its recently discovered replication into the vertebrate host does not necessarily imply a pathogenic role of this endosymbiont. A limit which characterizes tick parasitism in humans is represented by tick bite features: tick bites in humans often go unnoticed, because people may mistake the bite of another arthropod as that of a tick and components of tick saliva have anesthetic properties which inhibit host pain/hitch responses. Additionally, the predominant developmental stage of I. ricinus found to feed on humans are nymphs, which are difficult to detect due to their small size. For this reason, given the current lack of a reliable test for I. ricinus bite, the development of a diagnostic test for tick bite would be essential not only for the study of the clinical and epidemiological role of this vector, but also to evaluate the hypothesis of the tick bite as a risk factor for the onset of chronic degenerative diseases (Amyotrophic lateral sclerosis, rheumatologic and cardiovascular diseases). Moreover, the emergence of new tick-borne diseases and the re-emergence of existing ones, together with the rising dispersal of I. ricinus, increase the importance of this public health issue. The present PhD project has been focused on five principal researches: i) investigation of M. mitochondrii presence in blood and sera of roe deer (Capreolus capreolus) individuals through molecular and serological analyses; ii) serological detection of anti-M. mitochondrii antibodies in humans suffering from symptoms referable to tick-borne diseases from several European countries to investigate whether a protein of M. mitochondrii (FliD) could represent a marker suitable for diagnosis of I. ricinus bite; iii) study of the time and kinetic of seroconversion against the flagellar FliD protein of M. mitochondrii and salivary glands crude protein extract of I. ricinus ticks in rabbits experimentally infested with this tick species; iv) detection of additional antigenic proteins belonging to I. ricinus and M. mitochondrii to use as potential biomarkers for tick bite in a peptide-based ELISA assay; v) determination of the genetic variability of a previously undescribed Midichloria strain found in African Hyalomma marginatum ticks collected from migratory birds on Ventotene Island (Central Italy) and the quantification of the amount of this bacterium in ticks using a novel Real Time approach. Here is presented a brief description of each performed research: i) M. mitochondrii is a tick-borne intracellular bacterium of the order Rickettsiales, found with high prevalence in I. ricinus. M. mitochondrii is capable of vertical transmission in the tick, but recently evidence of potential horizontal transmission to the tick hosts through the blood meal has been reported. We investigated the presence of the bacterium in the blood of roe deer (C. capreolus) collected from an area known to be highly infested with I. ricinus ticks. We collected blood and sera samples for 3 yr in Gardouch (Haute Garonne, France) and subjected them to molecular screening through PCR and to serological investigation using enzyme-linked immunosorbent assay and Western blot. Bacterial DNA was detected in the blood of four of seven animals, but only at one or two points in time, whereas all sera were positive for M. mitochondrii antigens at all times. Our results indicated that the presence of the bacterium in the blood is transient, but the antibody response appeared to be long-lasting, possibly due to constant exposure to tick bites, and thus to repeated injection of bacteria. The role of M. mitochondrii in the mammalian host, and its interaction with other tick-borne bacteria, remains unknown. ii) The most common tick species parasitizing humans in Europe is I. ricinus, the main vector of the causative agent of Lyme disease B. burgdorferi in this continent. This tick species also hosts the endosymbiont M. mitochondrii, which can be transmitted to the vertebrate host during the tick blood meal. Since a high proportion of tick bites remains unnoticed due to rarity of immediate symptoms, and a reliable test for I. ricinus bite is still lacking, we investigated whether seropositivity toward a protein of M. mitochondrii (rFliD) could represent a marker suitable for diagnosis of I. ricinus bite in humans, focusing on samples collected in several European countries at different risk of tick bite. We screened 274 sera collected from patients suffering from different symptoms potentially referable to tick-borne diseases using an ELISA protocol to detect anti-M. mitochondrii antibodies. In the cohort of examined patients, the majority (179) had been collected from patients who remembered a tick bite, the remaining (95) from patients that did not recall a tick bite. Our results show a clear trend indicating that positivity to M. mitochondrii is higher where tick bite can be regarded as certain/almost certain, and lower where there is an uncertainty on the bite, with the highest positivity in Lyme patients (47.3%) and the lowest (2%) in negative controls. According to the obtained results, M. mitochondrii can be regarded as a useful source of antigens for the development of a serological test for I. ricinus bite. In prospect, additional antigens from M. mitochondrii and from the tick salivary glands could be incorporated in a multi-antigen test for the diagnosis of the tick bite. iii) I. ricinus endosymbiont M. mitochondrii, localized in ovaries and in salivary glands is vertically transmitted and is present in 100% of wild adult females, while prevalence values drop after some generations under laboratory conditions. Given the transmission of this bacterium to the vertebrate host during I. ricinus blood meal, this work was focused on monitoring M. mitochondrii antigens and DNA in a vertebrate model after infestation with I. ricinus for a time-span of four months. Two groups of rabbits were infested with I. ricinus females, respectively from the wild (naturally infected with the symbiont) and laboratory strain (lab; considered devoid of M. mitochondrii after quantitative PCR investigations) and screened using molecular and serological assays at nine time points. M. mitochondrii presence was detected in rabbits infested with wild I. ricinus ticks, but surprisingly also in those infested with lab ticks, albeit at later time points. This result prompted a more sensitive molecular screening of lab ticks, which were found to harbor very low symbiont loads. Our results indicate that transmission of the bacterium occurs even at low bacterial loads, and that antibody response against M. mitochondrii antigens begins within one week post-infestation with wild I. ricinus. Circulating DNA was detected in the blood of rabbits belonging to both groups up to the end of the experiment, suggesting a replication of the symbiont inside the vertebrate host. iv) I. ricinus bite could represent a risk factor for the onset of several chronic-degenerative diseases (amyotrophic lateral sclerosis, myocardiopathy, and multiple sclerosis). This hypothesis opens the necessity to obtain an effective test capable of identifying bites of this tick species in human and animal populations. The limits of the tick parasitism (misinformation in human population, features of the tick bite such as absence of itch, pain and immediate symptoms) prompted us to detect antigenic proteins belonging to I. ricinus salivary glands and to its endosymbiont M. mitochondrii to use as markers for tick bite. Additional antigenic proteins have been detected after immunoblotting analyses using protein extracts of salivary glands collected from I. ricinus adult females, and sera samples collected from experimentally infested rabbits. Novel peptides were designed from the most antigenic epitopes of each protein identified to detect antigenic markers for I. ricinus bite through an ELISA assay. The aim of the present work was to evaluate the occurrence of I. ricinus bite in different vertebrate hosts (rabbits, humans and roe deer) testing, through an ELISA approach, selected peptides developed from the identification of I. ricinus and M. mitochondrii antigenic proteins. The percentage of positivity, albeit low, found in tested human sera collected from subjects parasitized by ticks in Europe, and found all negative to M. mitochondrii, contribute to increase the percentage of sensitivity to investigate tick exposure. Interestingly, results obtained in rabbit sera testing two peptides showed positivity up to 16 weeks after experimental infestation, reinforcing the good property of these peptides as tick exposure indicator, with the final aim to combine different peptides in a multi-antigen ELISA test as diagnostic assay for tick bite to enhance the sensitivity of the test. v) Ticks can be easily transported over long-distances while feeding in their hosts, in particular avian species may contribute to tick dispersal across geographical barriers during migration. The role of migrating birds in the spread of ticks has epidemiological implications, since birds may contribute to the dispersal of tick-borne pathogens and microorganisms. Although the high number of actual pathogens that ticks can host, especially concerning the Ixodidae family (“hard ticks”), a variety of symbiotic bacteria have been detected including members of the Rickettsiaceae, Anaplasmataceae and Midichloriaceae families (order Rickettsiales). Since many bird species are common hosts of tick species harboring bacteria belonging to Midichloriaceae family (i.e. Ixodes and Hyalomma tick genera), in this work we determined the existence of a previously undescribed Midichloria bacteria strain carried by African H. marginatum nymphs collected on Italian Island of Ventotene from migratory birds travelling back from Africa. Additionally, we quantified through an innovative Real time PCR approach this bacterium in H. marginatum nymphs showing a higher bacteria load after the blood meal, similarly to what has been observed for the symbiont M. mitochondrii in Ixodes ricinus ticks. Future studies should investigate the presence of this Midichloria bacterium in adult H. marginatum ticks, in order to increase the knowledge concerning its role in the biology of this tick species and its transmission mode during the tick bite.