Background: Rodents are considered to contribute strongly to the risk of tick-borne diseases by feedingIxodes ricinuslarvae and by acting as amplifying hosts for pathogens. Here, we tested to what extent these two processes depend on rodent density, and for which pathogen species rodents synergistically contribute to the local disease risk, i.e. the density of infected nymphs (DIN).Methods: In a natural woodland, we manipulated rodent densities in plots of 2500 m²by either supplementing a critical food source (acorns) or by removing rodents during two years. Untreated plots were used as controls. Collected nymphs and rodent ear biopsies were tested for the presence of seven tick-borne microorganisms. Linear models were used to capture associations between rodents, nymphs, and pathogens.Results: Investigation of data from all plots, irrespective of the treatment, revealed a strong positive association between rodent density and nymphal density, nymphal infection prevalence (NIP) withBorrelia afzeliiandNeoehrlichia mikurensis, and hence DIN’s of these pathogens in the following year. The NIP, but not the DIN, of the bird-associatedBorrelia garinii, decreased with increasing rodent density. The NIPs ofBorrelia miyamotoiandRickettsia helveticawere independent of rodent density, and increasing rodent density moderately increased the DINs. In addition, NIPs ofBabesia microtiandSpiroplasma ixodetisdecreased with increasing rodent density, which had a non-linear association with DINs of these microorganisms.Conclusions: A positive density dependence for all rodent- and tick-associated tick-borne pathogens was found, despite the observation that some of them decreased in prevalence. The effects on the DINs were variable among microorganisms, more than likely due to contrasts in their biology (including transmission modes, host specificity and transmission efficiency). The strongest associations were found in rodent-associated pathogens that most heavily rely on horizontal transmission. Our results draw attention to the importance of considering transmission mode of a pathogen while developing preventative measures to successfully reduce the burden of disease.

Background: Ixodes ricinus ticks vector pathogens that cause serious health concerns. Like in other arthropods, the microbiome may affect the tick’s biology, with consequences for pathogen transmission. Here, we explored the bacterial communities of I. ricinus across its developmental stages and six geographic locations by the 16S rRNA amplicon sequencing, combined with quantification of the bacterial load. Results: A wide range of bacterial loads was found. Accurate quantification of low microbial biomass samples permitted comparisons to high biomass samples, despite the presence of contaminating DNA. The bacterial communities of ticks were associated with geographical location rather than life stage, and differences in Rickettsia abundance determined this association. Subsequently, we explored the geographical distribution of four vertically transmitted symbionts identified in the microbiome analysis. For that, we screened 16,555 nymphs from 19 forest sites for R. helvetica, Rickettsiella spp., Midichloria mitochondrii, and Spiroplasma ixodetis. Also, the infection rates and distributions of these symbionts were compared to the horizontally transmitted pathogens Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, and Neoehrlichia mikurensis. The infection rates of all vertically transmitted symbionts differed between the study sites, and none of the symbionts was present in all tested ticks suggesting a facultative association with I. ricinus. The proportions in which symbionts occurred in populations of I. ricinus were highly variable, but geographically close study sites expressed similar proportions. These patterns were in contrast to what we observed for horizontally transmitted pathogens. Lastly, nearly 12% of tested nymphs were free of any targeted microorganisms, which is in line with the microbiome analyses. Conclusions: Our results show that the microbiome of I. ricinus is highly variable, but changes gradually and ticks originating from geographically close forest sites express similar bacterial communities. This suggests that geography-related factors affect the infection rates of vertically transmitted symbionts in I. ricinus. Since some symbionts, such as R. helvetica can cause disease in humans, we propose that public health investigations consider geographical differences in its infection rates.

Human babesiosis in Europe has been attributed to infection with Babesia divergens and, to a lesser extent, with Babesia venatorum and Babesia microti, which are all transmitted to humans through a bite of Ixodes ricinus. These Babesia species circulate in the Netherlands, but autochthonous human babesiosis cases have not been reported so far. To gain more insight into the natural sources of these Babesia species, their presence in reservoir hosts and in I. ricinus was examined. Moreover, part of the ticks were tested for co-infections with other tick borne pathogens. In a cross-sectional study, qPCR-detection was used to determine the presence of Babesia species in 4611 tissue samples from 27 mammalian species and 13 bird species. Reverse line blotting (RLB) and qPCR detection of Babesia species were used to test 25,849 questing I. ricinus. Fragments of the 18S rDNA and cytochrome c oxidase subunit I (COI) gene from PCR-positive isolates were sequenced for confirmation and species identification and species-specific PCR reactions were performed on samples with suspected mixed infections. Babesia microti was found in two widespread rodent species: Myodes glareolus and Apodemus sylvaticus, whereas B. divergens was detected in the geographically restricted Cervus elaphus and Bison bonasus, and occasionally in free-ranging Ovis aries. B. venatorum was detected in the ubiquitous Capreolus capreolus, and occasionally in free-ranging O. aries. Species-specific PCR revealed co-infections in C. capreolus and C. elaphus, resulting in higher prevalence of B. venatorum and B. divergens than disclosed by qPCR detection, followed by 18S rDNA and COI sequencing. The non-zoonotic Babesia species found were Babesia capreoli, Babesia vulpes, Babesia sp. deer clade, and badger-associated Babesia species. The infection rate of zoonotic Babesia species in questing I. ricinus ticks was higher for Babesia clade I (2.6%) than Babesia clade X (1.9%). Co-infection of B. microti with Borrelia burgdorferi sensu lato and Neoehrlichia mikurensis in questing nymphs occurred more than expected, which reflects their mutual reservoir hosts, and suggests the possibility of co-transmission of these three pathogens to humans during a tick bite. The ubiquitous spread and abundance of B. microti and B. venatorum in their reservoir hosts and questing ticks imply some level of human exposure through tick bites. The restricted distribution of the wild reservoir hosts for B. divergens and its low infection rate in ticks might contribute to the absence of reported autochthonous cases of human babesiosis in the Netherlands.

Background: Microbial communities can affect disease risk by interfering with the transmission or maintenance of pathogens in blood-feeding arthropods. Here, we investigated whether bacterial communities vary between Ixodes ricinus nymphs which were or were not infected with horizontally transmitted human pathogens. Methods: Ticks from eight forest sites were tested for the presence of Borrelia burgdorferi sensu lato, Babesia spp., Anaplasma phagocytophilum, and Neoehrlichia mikurensis by quantitative polymerase chain reaction (qPCR), and their microbiomes were determined by 16S rRNA amplicon sequencing. Tick bacterial communities clustered poorly by pathogen infection status but better by geography. As a second approach, we analysed variation in tick microorganism community structure (in terms of species co-infection) across space using hierarchical modelling of species communities. For that, we analysed almost 14,000 nymphs, which were tested for the presence of horizontally transmitted pathogens B. burgdorferi s.l., A. phagocytophilum, and N. mikurensis, and the vertically transmitted tick symbionts Rickettsia helvetica, Rickettsiella spp., Spiroplasma ixodetis, and CandidatusMidichloria mitochondrii. Results: With the exception of Rickettsiella spp., all microorganisms had either significant negative (R. helvetica and A. phagocytophilum) or positive (S. ixodetis, N. mikurensis, and B. burgdorferi s.l.) associations with M. mitochondrii. Two tick symbionts, R. helvetica and S. ixodetis, were negatively associated with each other. As expected, both B. burgdorferi s.l. and N. mikurensis had a significant positive association with each other and a negative association with A. phagocytophilum. Although these few specific associations do not appear to have a large effect on the entire microbiome composition, they can still be relevant for tick-borne pathogen dynamics. Conclusions: Based on our results, we propose that M. mitochondrii alters the propensity of ticks to acquire or maintain horizontally acquired pathogens. The underlying mechanisms for some of these remarkable interactions are discussed herein and merit further investigation. Graphical

Rickettsiella species are bacterial symbionts that are present in a great variety of arthropod species, including ixodid ticks. However, little is known about their genetic diversity and distribution in Ixodes ricinus, as well as their relationship with other tick-associated bacteria. In this study, we investigated the occurrence and the genetic diversity of Rickettsiella spp. in I. ricinus throughout Europe and evaluated any preferential and antagonistic associations with Candidatus Midichloria mitochondrii and the pathogens Borrelia burgdorferi sensu lato and Borrelia miyamotoi. Rickettsiella spp. were detected in most I. ricinus populations investigated, encompassing a wide array of climate types and environments. The infection prevalence significantly differed between geographic locations and was significantly higher in adults than in immature life stages. Phylogenetic investigations and protein characterization disclosed four Rickettsiella clades (I–IV). Close phylogenetic relations were observed between Rickettsiella strains of I. ricinus and other arthropod species. Isolation patterns were detected for Clades II and IV, which were restricted to specific geographic areas. Lastly, although coinfections occurred, we did not detect significant associations between Rickettsiella spp. and the other tick-associated bacteria investigated. Our results suggest that Rickettsiella spp. are a genetically and biologically diverse facultative symbiont of I. ricinus and that their distribution among tick populations could be influenced by environmental components.

Borrelia miyamotoi is a spirochete transmitted by several ixodid tick species. It causes a relapsing fever in humans and is currently considered as an emerging pathogen. In Europe, B. miyamotoi seems to occur at low prevalence in Ixodes ricinus ticks but has a wide distribution. Here we report the first detection of B. miyamotoi in Ixodes ricinus ticks collected in two independent studies conducted in 2016 in the north-eastern and north-western Alps, Italy. Three out of 405 nymphs (0.74%) tested positive for Borrelia miyamotoi. In particular, B. miyamotoi was found in 2/365 nymphs in the western and in 1/40 nymphs in the eastern alpine area. These are the first findings of B. miyamotoi in Italy. Exposure to B. miyamotoi and risk of human infection may occur through tick bites in northern Italy. Relapsing fever caused by Borrelia miyamotoi has not yet been reported in Italy, but misdiagnoses with tick-borne encephalitis, human granulocytic anaplasmosis or other relapsing fever can occur. Our findings suggest that B. miyamotoi should be considered in the differential diagnosis of febrile patients originating from Lyme borreliosis endemic regions. The distribution of this pathogen and its relevance to public health need further investigation.