Dieter Heylen

Prevalence of Neoehrlichia mikurensis in ticks and rodents from North-west Europe.

BackgroundNeoehrlichia mikurensis s an emerging and vector-borne zoonosis: The first human disease cases were reported in 2010. Limited information is available about the prevalence and distribution of Neoehrlichia mikurensis in Europe, its natural life cycle and reservoir hosts. An Ehrlichia-like schotti variant has been described in questing Ixodes ricinus ticks, which could be identical to Neoehrlichia mikurensis.MethodsThree genetic markers, 16S rDNA, gltA and GroEL, of Ehrlichia schotti-positive tick lysates were amplified, sequenced and compared to sequences from Neoehrlichia mikurensis. Based on these DNA sequences, a multiplex real-time PCR was developed to specifically detect Neoehrlichia mikurensis in combination with Anaplasma phagocytophilum in tick lysates. Various tick species from different life-stages, particularly Ixodes ricinus nymphs, were collected from the vegetation or wildlife. Tick lysates and DNA derived from organs of wild rodents were tested by PCR-based methods for the presence of Neoehrlichia mikurensis. Prevalence of Neoehrlichia mikurensis was calculated together with confidence intervals using Fishers exact test.ResultsThe three genetic markers of Ehrlichia schotti-positive field isolates were similar or identical to Neoehrlichia mikurensis. Neoehrlichia mikurensis was found to be ubiquitously spread in the Netherlands and Belgium, but was not detected in the 401 tick samples from the UK. Neoehrlichia mikurensis was found in nymphs and adult Ixodes ricinus ticks, but neither in their larvae, nor in any other tick species tested. Neoehrlichia mikurensis was detected in diverse organs of some rodent species. Engorging ticks from red deer, European mouflon, wild boar and sheep were found positive for Neoehrlichia mikurensis.ConclusionsEhrlichia schotti is similar, if not identical, to Neoehrlichia mikurensis. Neoehrlichia mikurensis is present in questing Ixodes ricinus ticks throughout the Netherlands and Belgium. We propose that Ixodes ricinus can transstadially, but not transovarially, transmit this microorganism, and that different rodent species may act as reservoir hosts. These data further imply that wildlife and humans are frequently exposed to Neoehrlichia mikurensis- infected ticks through tick bites. Future studies should aim to investigate to what extent Neoehrlichia mikurensis poses a risk to public health.

Circulation of four Anaplasma phagocytophilum ecotypes in Europe

BackgroundAnaplasma phagocytophilum is the etiological agent of granulocytic anaplasmosis in humans and animals. Wild animals and ticks play key roles in the enzootic cycles of the pathogen. Potential ecotypes of A. phagocytophilum have been characterized genetically, but their host range, zoonotic potential and transmission dynamics has only incompletely been resolved.MethodsThe presence of A. phagocytophilum DNA was determined in more than 6000 ixodid ticks collected from the vegetation and wildlife, in 289 tissue samples from wild and domestic animals, and 69 keds collected from deer, originating from various geographic locations in The Netherlands and Belgium. From the qPCR-positive lysates, a fragment of the groEL-gene was amplified and sequenced. Additional groEL sequences from ticks and animals from Europe were obtained from GenBank, and sequences from human cases were obtained through literature searches. Statistical analyses were performed to identify A. phagocytophilum ecotypes, to assess their host range and their zoonotic potential. The population dynamics of A. phagocytophilum ecotypes was investigated using population genetic analyses.ResultsDNA of A. phagocytophilum was present in all stages of questing and feeding Ixodes ricinus, feeding I. hexagonus, I. frontalis, I. trianguliceps, and deer keds, but was absent in questing I. arboricola and Dermacentor reticulatus. DNA of A. phagocytophilum was present in feeding ticks and tissues from many vertebrates, including roe deer, mouflon, red foxes, wild boar, sheep and hedgehogs but was rarely found in rodents and birds and was absent in badgers and lizards. Four geographically dispersed A. phagocytophilum ecotypes were identified, that had significantly different host ranges. All sequences from human cases belonged to only one of these ecotypes. Based on population genetic parameters, the potentially zoonotic ecotype showed significant expansion.ConclusionFour ecotypes of A. phagocytophilum with differential enzootic cycles were identified. So far, all human cases clustered in only one of these ecotypes. The zoonotic ecotype has the broadest range of wildlife hosts. The expansion of the zoonotic A. phagocytophilum ecotype indicates a recent increase of the acarological risk of exposure of humans and animals.

Transmission dynamics of Borrelia burgdorferi s.l. in a bird tick community

We examined the Borrelia burgdorferi sensu lato circulation in a tick community consisting of three species (Ixodes ricinus, I. frontalis, I. arboricola) with contrasting ecologies, but sharing two European songbird hosts (Parus major and Cyanistes caeruleus). Parus major had the highest infestation rates, primarily due to larger numbers of I. ricinus, and probably because of their greater low-level foraging. The prevalence of Borrelia in feeding ticks did not significantly differ between the two bird species; however, P. major in particular hosted large numbers of Borrelia-infected I. frontalis and I. ricinus larvae, suggesting that the species facilitates Borrelia transmission. The low but significant numbers of Borrelia in questing I. arboricola ticks also provides the first field data to suggest that it is competent in maintaining Borrelia. Aside from Borrelia garinii, a high number of less dominant genospecies was observed, including several mammalian genospecies and the first record of Borrelia turdi for North-Western Europe. Borrelia burgdorferi sensu lato IGS genotypes were shared between I. arboricola and I. ricinus and between I. frontalis and I. ricinus, but not between I. arboricola and I. frontalis. This suggests that the Borrelia spp. transmission cycles can be maintained by bird-specific ticks, and bridged by I. ricinus to other hosts outside bird-tick cycles.

Host races in Ixodes ricinus, the European vector of Lyme borreliosis

Ixodes ricinus is a European tick that transmits numerous pathogenic agents, including the bacteria that cause Lyme disease (some genospecies of Borrelia burgdorferi sensu lato complex). This tick has been considered as a classic example of an extreme generalist vector. However, host-associations in such vector species are difficult to determine from field observations alone and recent work suggests that host specificity may be more frequent in ticks than previously thought. The presence of host-associated vector groups can significantly alter the circulation and evolutionary pathway of associated pathogens. In this paper, we explicitly test for host-associated genetic structure in I. ricinus. We analyzed genetic variability at 11 microsatellite markers in a large sample of ticks collected directly from trapped wild animals (birds, rodents, lizards, wild boar and roe deer) at five sites in Western and Central Europe. We found significant levels of genetic structure both among host individuals and among host types within local populations, suggesting that host use is not random in I. ricinus. These results help explain previous patterns of structure found in off-host tick samples, along with epidemiological observations of Lyme disease.

Lack of resistance against the tick Ixodes ricinus in two related passerine bird species

Although many wild bird species may act as reservoir hosts for tick-transmitted diseases and/or support long-distance dispersal of infected ticks, to date no research has been done on the extent to which songbirds may acquire resistance to ixodid ticks. Here we investigate whether two passerine species belonging to the family Paridae, the blue tit (Cyanistes caeruleus) and the great tit (Parus major), are able to acquire resistance after repeated infestations with Ixodes ricinus nymphs. As blue tits are less frequently exposed to I. ricinus in the wild than great tits, we expected I. ricinus to be less adapted towards the blue tits resistance mechanisms. Over the three infestation sessions we observed consistently high tick attachment rates and yields, high engorgement weights, and short engorgement and moulting durations, indicating that neither of the two songbird species is able to mount effective immune responses against I. ricinus nymphs after repeated infestations. As a consequence of the lack of resistance, birds were unable to prevent the direct harm (acute blood depletion) caused by tick feeding. Birds compensated the erythrocyte loss without reduction in general body condition (body mass corrected for tarsus length). The lack of resistance suggests that I. ricinus has a long co-evolutionary history with both avian hosts, which enables the tick to avoid or suppress the hosts resistance responses.

The design of complex sexual traits in male barn swallows: associations between signal attributes

Variation in the expression of sexually selected traits among individuals is widely investigated on the premise that these traits evolved to signal male quality. Significant repeatabilities of sexual signals and their associations with condition, mating success, survivorship and age may be the signatures of sexual selection. However, little is known about the relationship between these sexual attributes. Here we studied 28 acoustic and visual traits in the barn swallow, Hirundo rustica, that may potentially function in sexual selection. Based on effect sizes calculated at the between‐individual level, we assessed the relationship between repeatability, condition‐dependence, attractiveness, age‐dependence and viability indicator value of sexual traits using sexual signals as the units of analyses. Those traits that showed high within‐year repeatability also showed high between‐year repeatability, indicating that between‐individual variation is consistent within and among seasons. In addition, age‐dependence of traits, probably causing between‐year variation, was negatively related to between‐year repeatability. Condition‐dependence was negatively correlated with effect sizes for the extent to which traits predicted viability. Therefore, traits that are positively related to immediate condition are those that are negatively related to survival, which may be the signature of a trade‐off between current and future reproductive success ultimately reflecting signal reliability. No other significant relationship was found between trait attributes. We conclude that multiple sexual signals reflect different aspects of male quality in the barn swallow.

The Presence of Borrelia miyamotoi, A Relapsing Fever Spirochaete, in Questing Ixodes ricinus in Belgium and in The Netherlands

Borrelia miyamotoi is a tick‐borne bacterium that may cause relapsing fever in humans. As this pathogen has been discovered in Europe only recently, only little is known about its local impact on human health and its spatial distribution. In this study, we show the results of PCR screenings for B. miyamotoi in flagged Ixodes ricinus from Belgium and the Netherlands. B. miyamotoi was detected in nine of thirteen, and three of five locations from the Netherlands and Belgium, respectively. These outcomes indicate that B. miyamotoi is more spread than previously thought. The mean infection rate B. miyamotoi was 1.14% for Belgium and 3.84% for the Netherlands.

Spatial disaggregation of tick occurrence and ecology at a local scale as a preliminary step for spatial surveillance of tick-borne diseases: general framework and health implications in Belgium

BackgroundThe incidence of tick-borne diseases is increasing in Europe. Sub national information on tick distribution, ecology and vector status is often lacking. However, precise location of infection risk can lead to better targeted prevention measures, surveillance and control.MethodsIn this context, the current paper compiled geolocated tick occurrences in Belgium, a country where tick-borne disease has received little attention, in order to highlight the potential value of spatial approaches and draw some recommendations for future research priorities.ResultsMapping of 89,289 ticks over 654 sites revealed that ticks such as Ixodes ricinus and Ixodes hexagonus are largely present while Dermacentor reticulatus has a patchy distribution. Suspected hot spots of tick diversity might favor pathogen exchanges and suspected hot spots of I. ricinus abundance might increase human-vector contact locally. This underlines the necessity to map pathogens and ticks in detail. While I. ricinus is the main vector, I. hexagonus is a vector and reservoir of Borrelia burgdorferi s.l., which is active the whole year and is also found in urban settings. This and other nidiculous species bite humans less frequently, but seem to harbour pathogens. Their role in maintaining a pathogenic cycle within the wildlife merits investigation as they might facilitate transmission to humans if co-occurring with I. ricinus. Many micro-organisms are found abroad in tick species present in Belgium. Most have not been recorded locally but have not been searched for. Some are transmitted directly at the time of the bite, suggesting promotion of tick avoidance additionally to tick removal.ConclusionThis countrywide approach to tick-borne diseases has helped delineate recommendations for future research priorities necessary to design public health policies aimed at spatially integrating the major components of the ecological cycle of tick-borne diseases. A systematic survey of tick species and associated pathogens is called for in Europe, as well as better characterisation of species interaction in the ecology of tick-borne diseases, those being all tick species, pathogens, hosts and other species which might play a role in tick-borne diseases complex ecosystems.

Songbirds as general transmitters but selective amplifiers of Borrelia burgdorferi sensu lato genotypes in Ixodes rinicus ticks

We investigated to what extent a European songbird (Parus major) selectively transmits and amplifies Borrelia burgdorferi s.l. bacteria. Borrelia-naïve birds were recurrently exposed to Ixodes ricinus nymphs carrying a community of more than 34 5S-23S genotypes belonging to five genospecies (Borrelia garinii, Borrelia valaisiana, Borrelia afzelii, B. burgdorferi s.s. and Borrelia spielmanii). Fed ticks were screened for Borrelia after moulting. We found evidence for co-feeding transmission of avian and possibly also mammalian genotypes. Throughout the course of infestations, the infection rate of B. garinii and B. valaisiana increased, indicating successful amplification and transmission, while the infection rate for B. afzelii, B. burgdorferi s.s and B. spielmanii tended to decrease. Within the B. garinii and B. valaisiana genotype communities, certain genotypes were transmitted more than others. Moreover, birds were able to host mixed infections of B. garinii and B. valaisiana, as well as mixed infections of genotypes of the same genospecies. We experimentally show that resident songbirds transmit a broad range of Borrelia genotypes, but selectively amplify certain genotypes, and that one bird can transmit simultaneously several genotypes. Our results highlight the need to explicitly consider the association between genotypes and hosts, which may offer opportunities to point out which hosts are most responsible for the Borrelia presence in questing ticks.

Evolutionary changes in symbiont community structure in ticks

Ecological specialization to restricted diet niches is driven by obligate, and often maternally inherited, symbionts in many arthropod lineages. These heritable symbionts typically form evolutionarily stable associations with arthropods that can last for millions of years. Ticks were recently found to harbour such an obligate symbiont, Coxiella‐LE, that synthesizes B vitamins and cofactors not obtained in sufficient quantities from blood diet. In this study, the examination of 81 tick species shows that some Coxiella‐LE symbioses are evolutionarily stable with an ancient acquisition followed by codiversification as observed in ticks belonging to the Rhipicephalus genus. However, many other Coxiella‐LE symbioses are characterized by low evolutionary stability with frequent host shifts and extinction events. Further examination revealed the presence of nine other genera of maternally inherited bacteria in ticks. Although these nine symbionts were primarily thought to be facultative, their distribution among tick species rather suggests that at least four may have independently replaced Coxiella‐LE and likely represent alternative obligate symbionts. Phylogenetic evidence otherwise indicates that cocladogenesis is globally rare in these symbioses as most originate via horizontal transfer of an existing symbiont between unrelated tick species. As a result, the structure of these symbiont communities is not fixed and stable across the tick phylogeny. Most importantly, the symbiont communities commonly reach high levels of diversity with up to six unrelated maternally inherited bacteria coexisting within host species. We further conjecture that interactions among coexisting symbionts are pivotal drivers of community structure both among and within tick species.