Ixodes ricinus, commonly known as the castor bean tick and sheep tick, is a significant vector of various diseases, such as tick-borne encephalitis and Lyme borreliosis. Owing to climate change, the distribution and activity of I. ricinus are expected to increase, leading to an increase in the number of diseases transmitted by this species. Most distribution models and ecological niche models utilize macroclimate datasets such as WorldClim or CHELSA to map the distribution of disease-transmitting ticks. However, microclimatic factors are crucial for the activity and survival of small arthropods. In this study, an ecological niche modelling approach was used to assess the climatic suitability of I. ricinus using both microclimatic and macroclimatic parameters. A Mixed model was built by combining parameters from the Soiltemp (microclimate) and Wordclim (macroclimate) databases, whereas a Macroclimate model was built with the CHELSA dataset. Additionally, future suitabilities were projected via the macroclimate model under the SSP3-7.0 and SSP5-8.5 scenarios. Macroclimate and Mixed models showed similar distributions, confirming the current distribution of I. ricinus. The most important climatic factors were seasonality, annual temperature range, humidity and precipitation. Future projections suggest significant expansion in northern and eastern Europe, with notable declines in southern Europe.

In the present study, we identified the ectoparasite communities of red foxes in three regions of Poland that encompassed two endemic regions for the occurrence of Dermacentor reticulatus, as well as a region that is free of this tick species (‘gap’ area). Our study sites were selected to enable the role of foxes as hosts for juvenile (nest dwelling) and adult (exophilic) D. reticulatus ticks to be determined, and to assess their contribution to the spread of this important vector of Babesia canis. We compared also ectoparasite communities between adult foxes with those of fox cubs. Finally, we carried out a systematic search for subcutaneous ticks determining their prevalence and abundance. In 2016–2018, 366 adult foxes and 25 live-trapped cubs were examined for ectoparasites. Ectoparasites were identified based on morphological features, PCR amplification and sequencing. The total prevalence of ectoparasites was higher in cubs (68%) than in adults (62.8%). In adults, 15 parasite species were recorded, including four tick species, seven flea species, scabies, and one Anopluran species each in the genera Felicola and Lipoptena. In cubs, six ectoparasite species were found, including Ixodes kaiseri, a species not found in adults. Although Ixodes ricinus and D. reticulatus were the dominant tick species on adult foxes, no D. reticulatus ticks were found on cubs. Subcutaneous ticks were common (38%) and abundant in all areas. Molecular analysis of subcutaneous nodules allowed the identification of 17 I. ricinus and five D. reticulatus. In conclusion, red foxes play a minor role as hosts of D. reticulatus.

Apicomplexan haemoparasites of the genera Schellackia Reichenow, 1919, and Karyolysus Labbé, 1894, seem to be common in lizards and widespread across the world. For decades, their identification has been based on morphological descriptions and life cycle patterns, with molecular characterizations, applied only recently. We used molecular characterization to confirm the identification of haemoparasites detected by microscopy in blood smears of Lacerta schreiberi Bedriaga, 1878, a lizard of the Iberian Peninsula. Since blood samples other than blood smears were not available from the studied lizards, 264 engorged ticks Ixodes ricinus (Linneaus, 1758) collected from them were used as an alternative non-invasive source of haemoparasite DNA for molecular genetic analyses. Of the 48 blood smears microscopically examined, 31 were positive for blood parasites (64.6% prevalence). We identified trophozoites and gamonts similar to Karyolysus lacazei (Labbé, 1894) (24/48; 50%) and Schellackia-like sporozoites (20/48; 41.7%). Mixed infections with both species occurred in 13 blood smears (27.1%). Sequence data were obtained for both parasites from engorged ticks. Phylogenetic analyses placed our unique haemogregarine sequence within the Karyolysus clade, nevertheless, within substantial polytomy. Thus, according to its morphology and effect on the host cell, we refer to this haemogregarine as Karyolysus cf. lacazei. Besides the Schellackia sequences being identical to a previously identified haplotype, we also obtained sequences of three new closely related haplotypes.

The aim of this review paper is to review the data on tea tree (Melaleuca alternifolia) as an antimicrobial, antioxidant and acaricidal in poultry production. Tea tree exhibits a wide spectrum of antimicrobial activities with minimal inhibitory concentrations between 0.12 and 4 mg/ml. Its modes of action against Gram-negative bacterium Escherichia coli (0.12 to 1.5 mg/ml), Gram-positive bacterium Staphylococcus aureus (0.12 to 1 mg/ml), Aspergillus fumigatus (1.78 mg/ml) and yeast, Candida albicans (0.05 to 0.5 mg/ml) have been investigated using a range of different methods. As an antimicrobial, tea tree has high antifungal, bacteriostatic and germicidal activity (e.g. a decrease of 73.8% in Candida sp.), because of its components such as terpinen-4-ol, α-terpineol, linalool, α-pinene, β-pinene, β-myrcene and 1,8-cineole. Its bioactive compounds such as α-terpinene, α-terpinolene and γ-terpinene show high antioxidant activity when applied in concentrations of 100 and 200 µl/ml, while its essential oils demonstrated free radical scavenging activity of 60 to 80%. Tea trees insecticidal and acaricidal properties have been tested for tick control. The mortality of ticks (Ixodes ricinus) and poultry red mites (Dermanyssus gallinae) have been recorded at levels over 60% and 80%, respectively, when used in concentrations of 0.15 to 0.30 mg/cm2 during in vitro testing, and in vivo, when sprayed in poultry houses. When tea tree was used in a form of essential oils as a dietary supplement in concentrations of 50 to 150 mg/kg in broiler chicken diets, a significant increase in daily weight (by around 7%) and decrease in morbidity and mortality were seen. Additionally, when applied in laying hen nutrition, a significant increase in daily egg production has been recorded. Tea tree essential oils when supplemented in poultry diets have high positive effects regarding productivity performance, but this requires further field experiments to clarify standardisation of the material and effective inclusion levels.

Lyme disease is caused by bacteria of the Borrelia burgdorferi genospecies complex and transmitted by Ixodid ticks. In North America only one pathogenic genospecies occurs, in Europe there are several. According to the dilution effect hypothesis (DEH), formulated in North America, nymphal infection prevalence (NIP) decreases with increasing host diversity since host species differ in transmission potential. We analysed Borrelia infection in nymphs from 94 forest stands in Belgium, which are part of a diversification gradient with a supposedly related increasing host diversity: from pine stands without to oak stands with a shrub layer. We expected changing tree species and forest structure to increase host diversity and decrease NIP. In contrast with the DEH, NIP did not differ between different forest types. Genospecies diversity however, and presumably also host diversity, was higher in oak than in pine stands. Infected nymphs tended to harbour Borrelia afzelii infection more often in pine stands while Borrelia garinii and Borrelia burgdorferi ss. infection appeared to be more prevalent in oak stands. This has important health consequences, since the latter two cause more severe disease manifestations. We show that the DEH must be nuanced for Europe and should consider the response of multiple pathogenic genospecies.

This paper reports the first detection of Borrelia miyamotoi in UK Ixodes ricinus ticks. It also reports on the presence and infection rates of I. ricinus for a number of other tick-borne pathogens of public health importance. Ticks from seven regions in southern England were screened for B. miyamotoi, Borrelia burgdorferi sensu lato (s.l.), Anaplasma phagocytophilum and Neoehrlichia mikurensis using qPCR. A total of 954 I. ricinus ticks were tested, 40 were positive for B. burgdorferi s.l., 22 positive for A. phagocytophilum and three positive for B. miyamotoi, with no N. mikurensis detected. The three positive B. miyamotoi ticks came from three geographically distinct areas, suggesting a widespread distribution, and from two separate years, suggesting some degree of endemicity. Understanding the prevalence of Borrelia and other tick-borne pathogens in ticks is crucial for locating high-risk areas of disease transmission.

Bird-specific ticks do not infest humans and livestock, but these ticks often share their avian hosts with generalist ticks that do. Therefore, their feeding activity may have an impact on the transmission of pathogens outside bird–tick transmission cycles. Here we examined the seasonal feeding activity of the tree-hole tick (Ixodes arboricola) in relation to the activity of its hole-breeding hosts (Parus major and Cyanistes caeruleus). We analysed data on ticks derived from birds, on the abundance of engorged ticks inside nest boxes, and on bird nests that were experimentally exposed to ticks. We observed a non-random pattern of feeding associated with the tick instar and host age. The majority of adult ticks fed on nestlings, while nymphs and larvae fed on both free-flying birds and nestlings. Due to their fast development, some ticks were able to feed twice within the same breeding season. The highest infestation rates in free-flying birds were found during the pre-breeding period and during autumn and winter when birds roost inside cavities. Except during winter, feeding of I. arboricola overlapped in time with the generalist Ixodes ricinus, implying that tick-borne microorganisms that are maintained by I. arboricola and birds could be bridged by I. ricinus to other hosts.

Aggregation of parasites amongst hosts is important for the epidemiology of vector-borne diseases because hosts that support the majority of the vector population are responsible for the majority of pathogen transmission. Ixodes ricinus ticks transmit numerous pathogens of medical importance including Borrelia burgdorferi s.l. and tick-borne encephalitis virus. One transmission route involved is ‘co-feeding transmission’, where larvae become infected via feeding alongside infected nymphs. The aggregation of ticks on hosts leads to an increase in the number of larvae feeding alongside nymphs, increasing the transmission potential via this route. The basic reproduction number, R0, can be used to identify whether a pathogen will become established if introduced. In the current study we use previously published tick, and pathogen, specific data to parameterize an R0 model to investigate how the degree of aggregation of ticks on hosts affects pathogen persistence. The coincident aggregated distribution permitted the establishment of tick-borne encephalitis virus but did not influence whether B. burgdorferi s.l. became established. The relationship between the k-exponent of the negative binomial distribution and R0 was also defined. Therefore, the degree of aggregation of ticks on small mammal hosts has important implications for the risk to human health in a given area.

The mainstream forestry policy in many European countries is to convert coniferous plantations into (semi-natural) deciduous woodlands. However, woodlands are the main habitat for Ixodes ricinus ticks. Therefore, assessing to what extent tick abundance and infection with Borrelia spirochetes are affected by forest composition and structure is a prerequisite for effective prevention of Lyme borreliosis. We selected a total of 25 pine and oak stands, both with and without an abundant shrub layer, in northern Belgium and estimated tick abundance between April and October 2008–2010. Additionally, the presence of deer beds was used as an indicator of relative deer habitat use. Borrelia infections in questing nymphs were determined by polymerase chain reactions. The abundance of larvae, nymphs, and adults was higher in oak stands compared to pine stands and increased with increasing shrub cover, most likely due to differences in habitat use by the ticks' main hosts. Whereas tick abundance was markedly higher in structure-rich oak stands compared to homogeneous pine stands, the Borrelia infection rates in nymphs did not differ significantly. Our results indicate that conversion towards structure-rich deciduous forests might create more suitable tick habitats, but we were unable to detect an effect on the infection rate.

Ixodes ricinus horizontal movement within a humidity gradient and the influence of infection by Borrelia burgdorferi sensu lato (s.l.) on tick walking were investigated. Nymphs were placed within an arena containing a humidity gradient ranging from 45 to 95% relative humidity (RH). After 1 h of acclimation at 70% RH ticks were released so that they could either stay, or walk towards either the wet or the dry end. Their position was recorded 2 h post-release. Fat content was quantified and Borrelia infection was detected using real-time PCR and PCR followed by Reverse Line Blotting. Among the 1500 ticks tested, 29·85% were infected. More low-fat nymphs walked inside the arena than high-fat individuals. When nymphs walked, more low-fat ticks walked towards wetter than drier air, whereas more high-fat individuals walked towards drier than wetter air. Among high-fat nymphs, a lower proportion of Borrelia-infected ticks walked inside the arena compared to uninfected individuals, as though spirochetes manipulated their arthropod vector to stay. However, Borrelia infection had no effect on walking direction towards the dry or the wet end. Hence, it appears that I. ricinus nymphs walk horizontally over short distances within a humidity gradient depending on both energy resources and Borrelia infection.

The application of palladium nanoparticles as electron-dense markers for labeling in both transmission and scanning electron microscopy requires their conjugation to a specific protein. The conjugation protocol described here includes the dihydrolipoic acid (DHLA) capping of Pd nanoparticles (8 nm equivalent diameter) and their subsequent covalent attachment to functional protein molecules such as streptavidin, protein A, or avidin. The single-step reaction was mediated using the cross-linking agent ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). The final Pd conjugates were fully functional, as demonstrated by labeling of ultrathin resin sections of either bovine serum albumin or secretory granules of the salivary gland isolated from the partially fed female Ixodes ricinus tick. The results of bovine serum labeling were quantified, statistically evaluated, and compared with results obtained using commercially available gold particle conjugates (10 nm diameter). The highest values of labeling density were achieved using both streptavidin-Pd (106 ± 7 particles/μm2) and protein A-Au conjugates (130 ± 18 particles/μm2) compared to a commercial streptavidin-Au (66 ± 16 particles/μm2) and protein A-Pd conjugates (70 ± 11 particles/μm2). The concentrations of both DHLA and EDC, pH during conjugation, and finally thorough washing away of unbound proteins crucially influenced conjugation.

In the epidemiology of infectious diseases, the basic reproduction number, R0, 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 R0 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 R0 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 R0 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.

Objective. Expression of the gene encoding Der-p2 allergen-like protein in the castor bean tick Ixodes ricinus is induced by blood intake. Tick Der-p2 allergen-like protein belongs to a diverse family of ML proteins that includes major allergens of house dust mites, human MD-2 or similar proteins from Drosophila melanogaster. In ticks, genes encoding proteins belonging to the ML protein family were identified, but their protein products have not been characterized yet. Methods. A gene encoding tick Der-p2 allergen-like protein was amplified from cDNA of engorged I. ricinus female using the gene-specific primers designed on a basis of partial sequences of related allergen-like genes. The tissue and state specific patterns of expression of the gene were analysed. The IgE binding activity of the produced recombinant protein was studied by use of ELISA. Results. Analysis of the expression pattern showed that the gene encoding the tick Der-p2 allergen-like protein is strongly induced by the bloodmeal in gut and haemolymph throughout all tick developmental stages. Der-p2 allergen-like protein possesses a putative lipid-binding site, according to the comparisons with the related proteins. The ability of tick Der-p2 allergen-like protein to bind immunoglobulin E (IgE) was revealed. Discussion. The presence of a putative lipid-binding domain in Der-p2 allergen-like protein and its ability to interact with IgE might indicate the involvement of the protein in the tick's immune response.

In non-permanent parasites the separation from the host should take place in suitable habitats that allow the continuation of their life cycle. Furthermore, detachment strategies determine the parasites' dispersal capability, a characteristic on which epidemiological dynamics and the evolution of host specificity centre. In this study we experimentally investigate in the laboratory how 2 congeneric tick species, with contrasting habitat requirements, time detachment from one of their current songbird hosts (Parus major). Ixodes arboricola is a nidicolous tick, infesting bats and birds breeding or roosting in tree holes. Ixodes ricinus is a non-nidicolous generalist that parasitizes mammals, birds and even reptiles. We experimentally infested full-grown great tits, P. major, and found that I. arboricola detaches during the night, the moment when P. major sleeps in tree holes. In contrast, I. ricinus detaches during the day, the moment when birds are most active. In addition we found that all I. ricinus immatures left the birds within 5.5 days, while in I. arboricola the detachment time was long (up to 20 days) and highly variable. We discuss these findings with respect to their implications on the ticks' dispersal capability and host specificity.

Many vector-borne pathogens whose primary vectors are generalists, such as Ixodid ticks, can infect a wide range of host species and are often zoonotic. Understanding their transmission dynamics is important for the development of disease management programmes. Models exist to describe the transmission dynamics of such diseases, but are necessarily simplistic and generally limited by knowledge of vector population dynamics. They are typically deterministic SIR-type models, which predict disease dynamics in a single, non-spatial, closed patch. Here we explore the limitations of such a model of louping-ill virus dynamics by challenging it with novel field data. The model was only partially successful in predicting Ixodes ricinus density and louping-ill virus prevalence at 6 Scottish sites. We extend the existing multi-host model by forming a two-patch model, incorporating the impact of roaming hosts. This demonstrates that host movement may account for some of the discrepancies between the original model and empirical data. We conclude that insights into the dynamics of multi-host vector-borne pathogens can be gained by using a simple two-patch model. Potential improvements to the model, incorporating aspects of spatial and temporal heterogeneity, are outlined.

The population dynamics of tick-borne disease agents and in particular the mechanisms which influence their persistence are examined with reference to the flavivirus that causes louping-ill in red grouse and sheep. Pockets of infection cause heavy mortality and the infection probably persists as a consequence of immigration of susceptible hosts. Seroprevalence is positively associated with temporal variations in vectors per host, although variation between areas is associated with the abundance of mountain hares. The presence of alternative tick hosts, particularly large mammals, provides additional hosts for increasing tick abundance. Grouse alone can not support the vectors and the pathogen but both can persist when a non-viraemic mammalian host supports the tick population and a sufficiently high number of nymphs bite grouse. These alternative hosts may also amplify virus through non-viraemic transmission by the process of co-feeding, although the relative significance of this has yet to be determined. Another possible route of infection is through the ingestion of vectors when feeding or preening. Trans-ovarial transmission is a potentially important mechanism for virus persistence but has not been recorded with louping-ill and Ixodes ricinus. The influence of non-viraemic hosts, both in the multiplication of vectors and the amplification of virus through non-viraemic transmission are considered significant for virus persistence.

Free-living animals are usually inhabited by a community of parasitic species that can interact with each other and alter both host susceptibility and parasite transmission. In this study we tested the prediction that an increase in the gastrointestinal nematode Heligmosomoides polygyrus would increase the infestation of the tick Ixodes ricinus, in free-living yellow-necked mice, Apodemus flavicollis. An extensive cross-sectional trapping survey identified a negative relationship between H. polygyrus and I. ricinus counter to the prediction. An experimental reduction of the nematode infection through anthelmintic treatment resulted in an increase in tick infestation, suggesting that this negative association was one of cause and effect. Host characteristics (breeding condition and age) and habitat variables also contributed to affect tick infestation. While these results were counter to the prediction, they still support the hypothesis that interactions between parasite species can shape parasite community dynamics in natural systems. Laboratory models may act differently from natural populations and the mechanism generating the negative association is discussed.

The hard tick Ixodes ricinus (Ixodidae) is the sole animal thus far shown to harbour an intra-mitochondrial bacterium, which has recently been named Midichloria mitochondrii. The objectives of this work were (i) to screen ixodid ticks for Midichloria-related bacteria and (ii) to determine whether these bacteria exploit the intra-mitochondrial niche in other tick species. Our main goal was to discover further models of this peculiar form of symbiosis. We have thus performed a PCR screening for Midichloria-related bacteria in samples of ixodid ticks collected in Italy, North America and Iceland. A total of 7 newly examined species from 5 genera were found positive for bacteria closely related to M. mitochondrii. Samples of the tick species Rhipicephalus bursa, found positive in the PCR screening, were analysed with transmission electron microscopy, which revealed the presence of bacteria both in the cytoplasm and in the mitochondria of the oocytes. There is thus evidence that bacteria invade mitochondria in at least 2 tick species. Phylogenetic analysis on the bacterial 16S rRNA gene sequences generated from positive specimens revealed that the bacteria form a monophyletic group within the order Rickettsiales. The phylogeny of Midichloria symbionts and related bacteria does not appear completely congruent with the phylogeny of the hosts.

Although Ixodes ricinus ticks are mainly associated with woodland, they are also present in open habitat such as pastures. The distribution of nymphal I. ricinus was monitored by drag sampling the vegetation in May–June 2003 on 61 grazed permanent pastures for cattle located in central France. After selecting explanatory variables from among a set of 155, tick abundance was modelled on the perimeter of the pasture using a negative binomial model that took into account data overdispersion. An abundant tree layer at the perimeter of the pasture associated with a high humidity before sampling greatly enhanced the average number of captured I. ricinus nymphs. The presence of apple or cherry trees around the pasture perimeter, the presence of trees or bushes at the pasture edge, woodland around the pasture and a high number of I. ricinus nymphs in the nearest woodland to the pasture were also favourable to nymph abundance in the pasture. The study highlighted that woodland vegetation associated with humidity and the presence of attractive foraging areas for tick hosts around the pasture played a key role in the abundance of I. ricinus. Finally, the results raised the question of whether and how transfer of ticks between woodland and grazed pastures occurs.

Although Babesia divergens is the the principal confirmed zoonotic Babesia sp. in Europe, there are gaps in our knowledge of its biology and transmission by the tick Ixodes ricinus. In order to reproduce the part of the parasite cycle that occurs in the vector, an in vitro animal skin feeding technique on blood containing in vitro cultivated B. divergens was developed. Parasite DNA was detected in all samples of salivary glands of nymphs and adults that had fed on parasitized blood as larvae and nymphs, respectively, indicating acquisition as well as a transtadial persistence of B. divergens. PCR performed on eggs and larvae produced by females that had fed on parasitized blood demonstrated the existence of a transovarial transmission of the parasite. Gorging B. divergens infected larvae on non-infected gerbils showed persistance of the parasite over moulting into the resulting nymphs. These results indicate that the parasitic stages infective for the vector (i.e. the sexual stages) can be produced in vitro. To our knowledge, this is the first report of artificial feeding of I. ricinus via membrane as well as in vitro transmission of B. divergens to its vector. The opportunities offered by the use of such a transmission model of a pathogen by I. ricinus are discussed.