Quantitative microbial population study reveals geographical differences in bacterial symbionts of Ixodes ricinus.

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.

The Genetic Diversity of Rickettsiella Symbionts in Ixodes ricinus Throughout Europe.

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.

Circulation of Babesia Species and Their Exposure to Humans through Ixodes Ricinus.

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.

Tripartite Interactions among Ixodiphagus hookeri, Ixodes ricinus and Deer: Differential Interference with Transmission Cycles of Tick-Borne Pathogens.

For the development of sustainable control of tick-borne diseases, insight is needed in biological factors that affect tick populations. Here, the ecological interactions among Ixodiphagus hookeri, Ixodes ricinus, and two vertebrate species groups were investigated in relation to their effects on tick-borne disease risk. In 1129 questing ticks, I. hookeri DNA was detected more often in I. ricinus nymphs (4.4%) than in larvae (0.5%) and not in adults. Therefore, we determined the infestation rate of I. hookeri in nymphs from 19 forest sites, where vertebrate, tick, and tick-borne pathogen communities had been previously quantified. We found higher than expected co-occurrence rates of I. hookeri with deer-associated Anaplasma phagocytophilum, and lower than expected rates with rodent-associated Borrelia afzelii and Neoehrlichia mikurensis. The prevalence of I. hookeri in nymphs varied between 0% and 16% and was positively correlated with the encounter probability of ungulates and the densities of all life stages of I. ricinus. Lastly, we investigated the emergence of I. hookeri from artificially fed, field-collected nymphs. Adult wasps emerged from seven of the 172 fed nymphs. From these observations, we inferred that I. hookeri is parasitizing I. ricinus larvae that are feeding on deer, rather than on rodents or in the vegetation. Since I. hookeri populations depend on deer abundance, the main propagation host of I. ricinus, these wasps have no apparent effect on tick populations. The presence of I. hookeri may directly interfere with the transmission cycle of A. phagocytophilum, but not with that of B. afzelii or N. mikurensis.

The scale affects our view on the identification and distribution of microbial communities in ticks.

Ticks transmit the highest variety of pathogens impacting human and animal health worldwide. It is now well established that ticks also harbour a microbial complex of coexisting symbionts, commensals and pathogens. With the development of high throughput sequencing technologies, studies dealing with such diverse bacterial composition in tick considerably increased in the past years and revealed an unexpected microbial diversity. These data on diversity and composition of the tick microbes are increasingly available, giving crucial details on microbial communities in ticks and improving our knowledge on the tick microbial community. However, consensus is currently lacking as to which scales (tick organs, individual specimens or species, communities of ticks, populations adapted to particular environmental conditions, spatial and temporal scales) best facilitate characterizing microbial community composition of ticks and understanding the diverse relationships among tick-borne bacteria. Temporal or spatial scales have a clear influence on how we conduct ecological studies, interpret results, and understand interactions between organisms that build the microbiome. We consider that patterns apparent at one scale can collapse into noise when viewed from other scales, indicating that processes shaping tick microbiome have a continuum of variability that has not yet been captured. Based on available reports, this review demonstrates how much the concept of scale is crucial to be considered in tick microbial community studies to improve our knowledge on tick microbe ecology and pathogen/microbiota interactions.

Effect of rodent density on tick and tick-borne pathogen populations: consequences for infectious disease risk.

BACKGROUND: Rodents are considered to contribute strongly to the risk of tick-borne diseases by feeding Ixodes ricinus larvae 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 m2 by 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) with Borrelia afzelii and Neoehrlichia mikurensis, and hence DIN's of these pathogens in the following year. The NIP, but not the DIN, of the bird-associated Borrelia garinii, decreased with increasing rodent density. The NIPs of Borrelia miyamotoi and Rickettsia helvetica were independent of rodent density, and increasing rodent density moderately increased the DINs. In addition, NIPs of Babesia microti and Spiroplasma ixodetis decreased 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.

Role of mustelids in the life-cycle of ixodid ticks and transmission cycles of four tick-borne pathogens.

BACKGROUND: Elucidating which wildlife species significantly contribute to the maintenance of Ixodes ricinus populations and the enzootic cycles of the pathogens they transmit is imperative in understanding the driving forces behind the emergence of tick-borne diseases. Here, we aimed to quantify the relative contribution of four mustelid species in the life-cycles of I. ricinus and Borrelia burgdorferi (sensu lato) in forested areas and to investigate their role in the transmission of other tick-borne pathogens. Road-killed badgers, pine martens, stone martens and polecats were collected in Belgium and the Netherlands. Their organs and feeding ticks were tested for the presence of tick-borne pathogens. RESULTS: Ixodes hexagonus and I. ricinus were found on half of the screened animals (n = 637). Pine martens had the highest I. ricinus burden, whereas polecats had the highest I. hexagonus burden. We detected DNA from B. burgdorferi (s.l.) and Anaplasma phagocytophilum in organs of all four mustelid species (n = 789), and Neoehrlichia mikurensis DNA was detected in all species, except badgers. DNA from B. miyamotoi was not detected in any of the investigated mustelids. From the 15 larvae of I. ricinus feeding on pine martens (n = 44), only one was positive for B. miyamotoi DNA, and all tested negative for B. burgdorferi (s.l.), N. mikurensis and A. phagocytophilum. The two feeding larvae from the investigated polecats (n = 364) and stone martens (n = 39) were negative for all four pathogens. The infection rate of N. mikurensis was higher in feeding nymphs collected from mustelids compared to questing nymphs, but not for B. burgdorferi (s.l.), B. miyamotoi or A. phagocytophilum. CONCLUSIONS: Although all stages of I. ricinus can be found on badgers, polecats, pine and stone martens, their relative contribution to the life-cycle of I. ricinus in forested areas is less than 1%. Consequently, the relative contribution of mustelids to the enzootic cycles of I. ricinus-borne pathogens is negligible, despite the presence of these pathogens in organs and feeding ticks. Interestingly, all four mustelid species carried all stages of I. hexagonus, potentially maintaining enzootic cycles of this tick species apart from the cycle involving hedgehogs as main host species.

Eco-epidemiology of Novel Bartonella Genotypes from Parasitic Flies of Insectivorous Bats.

Bats are important zoonotic reservoirs for many pathogens worldwide. Although their highly specialized ectoparasites, bat flies (Diptera: Hippoboscoidea), can transmit Bartonella bacteria including human pathogens, their eco-epidemiology is unexplored. Here, we analyzed the prevalence and diversity of Bartonella strains sampled from 10 bat fly species from 14 European bat species. We found high prevalence of Bartonella spp. in most bat fly species with wide geographical distribution. Bat species explained most of the variance in Bartonella distribution with the highest prevalence of infected flies recorded in species living in dense groups exclusively in caves. Bat gender but not bat fly gender was also an important factor with the more mobile male bats giving more opportunity for the ectoparasites to access several host individuals. We detected high diversity of Bartonella strains (18 sequences, 7 genotypes, in 9 bat fly species) comparable with tropical assemblages of bat-bat fly association. Most genotypes are novel (15 out of 18 recorded strains have a similarity of 92-99%, with three sequences having 100% similarity to Bartonella spp. sequences deposited in GenBank) with currently unknown pathogenicity; however, 4 of these sequences are similar (up to 92% sequence similarity) to Bartonella spp. with known zoonotic potential. The high prevalence and diversity of Bartonella spp. suggests a long shared evolution of these bacteria with bat flies and bats providing excellent study targets for the eco-epidemiology of host-vector-pathogen cycles.

Ticks and Borrelia in urban and peri-urban green space habitats in a city in southern England.

Ticks are becoming increasingly recognised as important vectors of pathogens in urban and peri-urban areas, including green space used for recreational activities. In the UK, the risk posed by ticks in such areas is largely unknown. In order to begin to assess the risk of ticks in urban/peri-urban areas in southern England, questing ticks were collected from five different habitat types (grassland, hedge, park, woodland and woodland edge) in a city during the spring, summer and autumn of 2013/2014 and screened for Borrelia burgdorferi sensu lato. In addition, seasonal differences in B. burgdorferi s.l. prevalence were also investigated at a single site during 2015. Ixodes ricinus presence and activity were significantly higher in woodland edge habitat and during spring surveys. DNA of Borrelia burgdorferi s.l. was detected in 18.1% of nymphs collected across the 25 sites during 2013 and 2014 and two nymphs also tested positive for the newly emerging tick-borne pathogen B. miyamotoi. Borrelia burgdorferi s.l. prevalence at a single site surveyed in 2015 were found to be significantly higher during spring and summer than in autumn, with B. garinii and B. valaisiana most commonly detected. These data indicate that a range of habitats within an urban area in southern England support ticks and that urban Borrelia transmission cycles may exist in some of the urban green spaces included in this study. Sites surveyed were frequently used by humans for recreational activities, providing opportunity for exposure to Borrelia infected ticks in an urban/peri-urban space that might not be typically associated with tick-borne disease transmission.

Presence of zoonotic agents in engorged ticks and hedgehog faeces from Erinaceus europaeus in (sub) urban areas.

BACKGROUND: European hedgehogs (Erinaceus europaeus) are hosts for Ixodes hexagonus and I. ricinus ticks, which are vectors for zoonotic microorganisms. In addition, hedgehogs may carry several enteric zoonoses as well. It is unclear to what extent a presence of pathogens in hedgehogs poses a risk to public health, as information on the presence of zoonotic agents in hedgehogs in urban areas is relatively scarce. METHODS: Engorged ticks and hedgehog faeces were collected from rehabilitating hedgehogs. Ticks were screened individually for presence of Borrelia burgdorferi sensu lato, B. miyamotoi, Anaplasma phagocytophilum, and Candidatus Neoehrlichia mikurensis using PCR-based assays. Faecal samples were screened for presence of Campylobacter, Salmonella, Giardia, Cryptosporidium, and extended-spectrum cephalosporin-resistant-Escherichia coli (ESC)-resistant E. coli, using both culture-based and PCR-based methods. RESULTS: Anaplasma phagocytophilum and Borrelia genospecies B. afzelii, B. spielmanii, B. garinii, and B. burgdorferi sensu stricto were detected in both I. hexagonus and I. ricinus ticks. Despite their widespread distribution in the Netherlands, B. miyamotoi and Candidatus N. mikurensis were not detected in collected ticks. Analysis of hedgehog faecal samples revealed the presence of Salmonella enterica subspecies enterica and Campylobacter jejuni. In addition, ESC-resistant E. coli were observed in high prevalence in faecal samples, but no Shiga-toxin producing-E.coli were detected. Finally, potentially zoonotic protozoan parasites were observed in hedgehog faecal samples as well, including Giardia duodenalis assemblage A, Cryptosporidium parvum subtypes IIaA17G1R1 and IIcA5G3, and C. hominis subtype IbA10G2. CONCLUSIONS: European hedgehogs in (sub)urban areas harbor a number of zoonotic agents, and therefore may contribute to the spread and transmission of zoonotic diseases. The relatively high prevalence of B. burgdorferi s.l. and A. phagocytophilum in engorged ticks, suggests that hedgehogs contribute to their enzootic cycles in (sub)urban areas. To what extent can hedgehogs maintain the enteric zoonotic agents in natural cycles, and the role of (spill-back from) humans remains to be investigated.