Ixodes ricinus (Ixodidae), an occasional phoront on necrophagous and coprophagous beetles in Europe

For ticks, phoretic behaviour using insects associated with vertebrates might offer an alternative strategy to host-seeking. Here we report for the first time the presence of immature stages of the most widespread tick species in Western Europe, Ixodes ricinus (Acari: Ixodidae), on three beetle species belonging to families Silphidae and Geotrupidae (Coleoptera). Specimens were collected while performing fieldwork surveys on insect diversity during the peak of tick’s questing behaviour, therefore, in July and August of 2009 and 2010. The collections took place in two Natural Parks, the Aiako Harria, Guipúzcoa in Northern Spain and Wellington Country Park, Berkshire, in England. The silphid species Nicrophorus vespilloides, together with the geotrupid Trypocopris pyrenaeus were both collected from pig-carcasses and both carried nymphs of I. ricinus; while, the geotrupid Anoplotrupes stercorosus was carrying a tick larva while feeding on Red deer dung. These findings revealed an unnoticed but common relation of ticks not only with decomposed animals but also with insect scavengers. We discuss the rationale of this phenomenon.

Presence of potentially pathogenic Babesia sp. for human in Ixodes ricinus in Switzerland

We have designed and performed a new PCR method based on the 18S rRNA in order to individuate the presence and the identity of Babesia parasites. Out of 1159 Ixodes ricinus (Acari: Ixodidae) ticks collected in four areas of Switzerland, nine were found to contain Babesia DNA. Sequencing of the short amplicon obtained (411-452 bp) allowed the identification of three human pathogenic species: Babesia microti, B. divergens, for the first time in Switzerland, Babesia sp. EU1. We also report coinfections with B. sp. EU1-Borrelia burgdorferi sensu stricto and Babesia sp. EU1-B. afzelii.

Distribution of the Ixodes ricinus-like ticks of eastern North America.

We analyzed the geographic distribution of the Ixodes ricinus-like ticks in eastern North America by comparing the mitochondrial 16S rDNA sequences of specimens sampled directly from the field during the 1990s. Two distinct lineages are evident. The southern clade includes ticks from the southeastern and middle-eastern regions of the United States. The range of the northern clade, which appears to have been restricted to the northeastern region until the mid-1900s, now extends throughout the northeastern and middle-eastern regions. These phyletic units correspond to northern and southern taxa that have previously been assigned specific status as Ixodes dammini and Ixodes scapularis, respectively. The expanding range of I. dammini appears to drive the present outbreaks of zoonotic disease in eastern North America that include Lyme disease and human babesiosis.

The paradox of the binomial Ixodes ricinus activity and the observed unimodal Lyme borreliosis season in Hungary

The change of ambient temperature plays a key role in determining the run of the annual Lyme season. Our aim was to explain the apparent contradiction between the annual unimodal Lyme borreliosis incidence and the bimodal Ixodes ricinus tick activity run – both observed in Hungary – by distinguishing the temperaturedependent seasonal human and tick activity, the temperature-independent factors, and the multiplicative effect of human outdoor activity in summer holiday, using data from Hungary in the period of 1998–2012. This separation was verified by modeling the Lyme incidence based on the separated factors, and comparing the run of the observed and modeled incidence. We demonstrated the bimodality of tick season by using the originally unimodal Lyme incidence data. To model the outdoor human activity, the amount of camping guest nights was used, which showed an irregular run from mid-June to September. The human outdoor activity showed a similar exponential correlation with ambient temperature to that what the relative incidence did. It was proved that summer holiday has great influence on Lyme incidence.

The paradox of the binomial Ixodes ricinus activity and the observed unimodal Lyme borreliosis season in Hungary

The change of ambient temperature plays a key role in determining the run of the annual Lyme season. Our aim was to explain the apparent contradiction between the annual unimodal Lyme borreliosis incidence and the bimodal Ixodes ricinus tick activity run – both observed in Hungary – by distinguishing the temperaturedependent seasonal human and tick activity, the temperature-independent factors, and the multiplicative effect of human outdoor activity in summer holiday, using data from Hungary in the period of 1998–2012. This separation was verified by modeling the Lyme incidence based on the separated factors, and comparing the run of the observed and modeled incidence. We demonstrated the bimodality of tick season by using the originally unimodal Lyme incidence data. To model the outdoor human activity, the amount of camping guest nights was used, which showed an irregular run from mid-June to September. The human outdoor activity showed a similar exponential correlation with ambient temperature to that what the relative incidence did. It was proved that summer holiday has great influence on Lyme incidence.

Ecological drivers of a vector borne pathogen: distribution and abundance of Borrelia burgdorferi sensu lato and its vector Ixodes ricinus in Scotland

Vector-borne disease emergence in recent decades has been associated with different environmental drivers including changes in habitat, hosts and climate. Lyme borreliosis is among the most important vector-borne diseases in the Northern hemisphere and is an emerging disease in Scotland. Transmitted by Ixodid tick vectors between large numbers of wild vertebrate host species, Lyme borreliosis is caused by bacteria from the Borrelia burgdorferi sensu lato species group. Ecological studies can inform how environmental factors such as host abundance and community composition, habitat and landscape heterogeneity contribute to spatial and temporal variation in risk from B. burgdorferi s.l. In this thesis a range of approaches were used to investigate the effects of vertebrate host communities and individual host species as drivers of B. burgdorferi s.l. dynamics and its tick vector Ixodes ricinus. Host species differ in reservoir competence for B. burgdorferi s.l. and as hosts for ticks. Deer are incompetent transmission hosts for B. burgdorferi s.l. but are significant hosts of all life-stages of I. ricinus. Rodents and birds are important transmission hosts of B. burgdorferi s.l. and common hosts of immature life-stages of I. ricinus. In this thesis, surveys of woodland sites revealed variable effects of deer density on B. burgdorferi prevalence, from no effect (Chapter 2) to a possible ‘dilution’ effect resulting in lower prevalence at higher deer densities (Chapter 3). An invasive species in Scotland, the grey squirrel (Sciurus carolinensis), was found to host diverse genotypes of B. burgdorferi s.l. and may act as a spill-over host for strains maintained by native host species (Chapter 4). Habitat fragmentation may alter the dynamics of B. burgdorferi s.l. via effects on the host community and host movements. In this thesis, there was lack of persistence of the rodent associated genospecies of B. burgdorferi s.l. within a naturally fragmented landscape (Chapter 3). Rodent host biology, particularly population cycles and dispersal ability are likely to affect pathogen persistence and recolonization in fragmented habitats. Heterogeneity in disease dynamics can occur spatially and temporally due to differences in the host community, habitat and climatic factors. Higher numbers of I. ricinus nymphs, and a higher probability of detecting a nymph infected with B. burgdorferi s.l., were found in areas with warmer climates estimated by growing degree days (Chapter 2). The ground vegetation type associated with the highest number of I. ricinus nymphs varied between studies in this thesis (Chapter 2 & 3) and does not appear to be a reliable predictor across large areas. B. burgdorferi s.l. prevalence and genospecies composition was highly variable for the same sites sampled in subsequent years (Chapter 2). This suggests that dynamic variables such as reservoir host densities and deer should be measured as well as more static habitat and climatic factors to understand the drivers of B. burgdorferi s.l. infection in ticks. Heterogeneity in parasite loads amongst hosts is a common finding which has implications for disease ecology and management. Using a 17-year data set for tick infestations in a wild bird community in Scotland, different effects of age and sex on tick burdens were found among four species of passerine bird (Chapter 5). There were also different rates of decline in tick burdens among bird species in response to a long term decrease in questing tick pressure over the study. Species specific patterns may be driven by differences in behaviour and immunity and highlight the importance of comparative approaches. Combining whole genome sequencing (WGS) and population genetics approaches offers a novel approach to identify ecological drivers of pathogen populations. An initial analysis of WGS from B. burgdorferi s.s. isolates sampled 16 years apart suggests that there is a signal of measurable evolution (Chapter 6). This suggests demographic analyses may be applied to understand ecological and evolutionary processes of these bacteria. This work shows how host communities, habitat and climatic factors can affect the local transmission dynamics of B. burgdorferi s.l. and the potential risk of infection to humans. Spatial and temporal heterogeneity in pathogen dynamics poses challenges for the prediction of risk. New tools such as WGS of the pathogen (Chapter 6) and blood meal analysis techniques will add power to future studies on the ecology and evolution of B. burgdorferi s.l.

Factors driving the abundance of ixodes ricinus ticks and the prevalence of zoonotic I. ricinus-borne pathogens in natural foci

Environmental factors may drive tick ecology and therefore tick-borne pathogen (TBP) epidemiology, which determines the risk to animals and humans of becoming infected by TBPs. For this reason, the aim of this study was to analyze the influence of environmental factors on the abundance of immature-stage Ixodes ricinus ticks and on the prevalence of two zoonotic I. ricinus-borne pathogens in natural foci of endemicity. I. ricinus abundance was measured at nine sites in the northern Iberian Peninsula by dragging the vegetation with a cotton flannelette, and ungulate abundance was measured by means of dung counts. In addition to ungulate abundance, data on variables related to spatial location, climate, and soil were gathered from the study sites. I. ricinus adults, nymphs, and larvae were collected from the vegetation, and a representative subsample of I. ricinus nymphs from each study site was analyzed by PCR for the detection of Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum DNA. Mean prevalences of these pathogens were 4.0% ± 1.8% and 20.5% ± 3.7%, respectively. Statistical analyses confirmed the influence of spatial factors, climate, and ungulate abundance on I. ricinus larva abundance, while nymph abundance was related only to climate. Interestingly, cattle abundance rather than deer abundance was the main driver of B. burgdorferi sensu lato and A. phagocytophilum prevalence in I. ricinus nymphs in the study sites, where both domestic and wild ungulates coexist. The increasing abundance of cattle seems to increase the risk of other hosts becoming infected by A. phagocytophilum, while reducing the risk of being infected by B. burgdorferi sensu lato. Controlling ticks in cattle in areas where they coexist with wild ungulates would be more effective for TBP control than reducing ungulate abundance.

Body mass and sex-biased parasitism in wood mice Apodemus sylvaticus

Male sex-biased parasitism (SBP) occurs across a range of mammalian taxa and two contrasting sets of hypotheses have been suggested for its establishment. The first invokes body size per se and suggests that larger individuals are either a larger target for parasites, trade off growth at the expense of immunity or cope better with parasitism than smaller individuals. The second suggests a sex-specific handicap whereby males have reduced immunocompetence compared to females due to the immunodepressive effects of testosterone. The current study investigated whether sex-biased parasitism is driven by host 'body size' or 'sex' using a rodent-tick (Apodemus sylvaticus-. Ixodes ricinus) system. Moreover, the presence or absence of large mammals at study sites were used to control the presence of immature ticks infesting wood mice, allowing the impacts of parasitism on host body mass and female reproduction to be assessed. As expected, male mice had greater tick loads than females and analyses suggested this sex-bias was driven by body mass as opposed to sex. It is therefore likely that larger individuals are a larger target for parasites, trade off growth at the expense of immunity or adapt behavioural responses to parasitism based on their body size. Parasite load had no effect on host body mass or female reproductive output suggesting individuals may alter behaviour or life history strategies to compensate for costs incurred through parasitism. Overall, this study lends support to the 'body size' hypothesis for the formation of sex-biased parasitism.

Investigating the persistence of tick-borne pathogens via the R-0 model

In the epidemiology of infectious diseases, the basic reproduction number, R-0, has a number of important applications, most notably it can be used to predict whether a pathogen is likely to become established, or persist, in a given area. We used the R-0 model to investigate the persistence of 3 tick-borne pathogens; Babesia microti, Anaplasma phagocytophilum and Borrelia burgdorferi sensu lato in an Apodemus sylvaticus-Ixodes ricinus system. The persistence of these pathogens was also determined empirically by screening questing ticks and wood mice by PCR. All 3 pathogens behaved differently in response to changes in the proportion of transmission hosts on which I. ricinus fed, the efficiency of transmission between the host and ticks and the abundance of larval and nymphal ticks found on small mammals. Empirical data supported theoretical predictions of the R-0 model. The transmission pathway employed and the duration of systemic infection were also identified as important factors responsible for establishment or persistence of tick-borne pathogens in a given tick-host system. The current study demonstrates how the R-0 model can be put to practical use to investigate factors affecting tick-borne pathogen persistence, which has important implications for animal and human health worldwide.

Non-hemagglutinating flaviviruses: molecular mechanisms for the emergence of new strains via adaptation to European ticks

Tick-borne encephalitis virus (TBEV) causes human epidemics across Eurasia. Clinical manifestations range from inapparent infections and fevers to fatal encephalitis but the factors that determine disease severity are currently undefined. TBEV is characteristically a hemagglutinating (HA) virus; the ability to agglutinate erythrocytes tentatively reflects virion receptor/fusion activity. However, for the past few years many atypical HA-deficient strains have been isolated from patients and also from the natural European host tick, Ixodes persulcatus. By analysing the sequences of HA-deficient strains we have identified 3 unique amino acid substitutions (D67G, E122G or D277A) in the envelope protein, each of which increases the net charge and hydrophobicity of the virion surface. Therefore, we genetically engineered virus mutants each containing one of these 3 substitutions; they all exhibited HA-deficiency. Unexpectedly, each genetically modified non-HA virus demonstrated increased TBEV reproduction in feeding Ixodes ricinus, not the recognised tick host for these strains. Moreover, virus transmission efficiency between infected and uninfected ticks co-feeding on mice was also intensified by each substitution. Retrospectively, the mutation D67G was identified in viruses isolated from patients with encephalitis. We propose that the emergence of atypical Siberian HA-deficient TBEV strains in Europe is linked to their molecular adaptation to local ticks. This process appears to be driven by the selection of single mutations that change the virion surface thus enhancing receptor/fusion function essential for TBEV entry into the unfamiliar tick species. As the consequence of this adaptive mutagenesis, some of these mutations also appear to enhance the ability of TBEV to cross the human blood-brain barrier, a likely explanation for fatal encephalitis. Future research will reveal if these emerging Siberian TBEV strains continue to disperse westwards across Europe by adaptation to the indigenous tick species and if they are associated with severe forms of TBE.

Identification of Bartonella species in rodents, shrews and cats in Denmark: detection of two B-henselae variants, one in cats and the other in the long-tailed field mouse

Small mammals and stray cats were trapped in two areas of North Zealand, Denmark, and their blood cultured for hemotrophic bacteria. Bacterial isolates were recovered in pure culture and subjected to 16S rDNA gene sequencing. Bartonella species were isolated from five mammalian species: B. grahamii from Microtus agrestis (field vole) and Apodemus flavicollis (yellow-necked field mouse); B. taylorii from M. agrestis, A. flavicollis and A. sylvaticus (long-tailed field mouse); B. tribocorum from A. flavicollis; R vinsonii subsp. vinsonii from M. agrestis and A. sylvaticus; and B. birtlesii from Sorex vulgaris (common shrew). In addition, two variant types of B. henselae were identified: variant I was recovered from three specimens of A. sylvaticus, and B. henselae variant 11 from I I cats; in each case this was the only B. henselae variant found. No Bartonella species was isolated from Clethrionomys glareolus (bank vole) or Micromys minutus (harvest mouse). These results suggest that B. henselae occurs in two animal reservoirs in this region, one of variant I in A. sylvaticus, which may be transmitted between mice by the tick Ixodes ricinus, and another of variant 11 in cats, which may be transmitted by the cat flea (Ctenocephalides felis). To our knowledge, this is the first report of the occurrence of B. henselae and B. tribocorum in Apodemus mice.