Spatial and Temporal Variability in Prevalence Rates of Members of the Borrelia burgdorferi Species Complex in Ixodes ricinus Ticks in Urban, Agricultural and Sylvatic Habitats in Slovakia.

Lyme borreliosis (LB) is the most prevalent tick-borne human infection in Europe, with increasing incidence during the latest decades. Abundant populations of Ixodes ricinus, the main vector of the causative agent, spirochetes from the Borrelia burgdorferi sensu lato (Bbsl) complex, have been observed in urban and suburban areas of Europe, in general, and Slovakia, particularly. Understanding the spread of infectious diseases is crucial for implementing effective control measures. Global changes affect contact rates of humans and animals with Borrelia-infected ticks and increase the risk of contracting LB. The aim of this study was to investigate spatial and temporal variation in prevalence of Bbsl and diversity of its species in questing I. ricinus from three sites representing urban/suburban, natural and agricultural habitat types in Slovakia. Ixodes ricinus nymphs and adults were collected by dragging the vegetation in green areas of Bratislava town (urban/suburban habitat), in the Small Carpathians Mountains (natural habitat) (south-western Slovakia) and in an agricultural habitat at Rozhanovce in eastern Slovakia. Borrelia presence in ticks was detected by PCR and Bbsl species were identified by restriction fragment length polymorphism (RFLP). Borrelia burgdorferi s.l. species in coinfected ticks were identified by reverse line blot. Significant spatial and temporal variability in prevalence of infected ticks was revealed in the explored habitats. The lowest total prevalence was detected in the urban/suburban habitat, whereas higher prevalence was found in the natural and agricultural habitat. Six Bbsl species were detected by RFLP in each habitat type -B. burgdorferi sensu stricto (s.s.), B. afzelii, B. garinii, B. valaisiana, B. lusitaniae and B. spielmanii. Coinfections accounted for 3% of the total infections, whereby B. kurtenbachii was identified by RLB and sequencing in mixed infection with B. burgdorferi s.s, B. garinii and B. valaisiana. This finding represents the first record of B. kurtenbachii in questing I. ricinus in Slovakia and Europe. Variations in the proportion of Bbsl species were found between nymphs and adults, between years and between habitat types. Spatial variations in prevalence patterns and proportion of Bbsl species were also confirmed between locations within a relatively short distance in the urban habitat. Habitat-related and spatial variations in Borrelia prevalence and distribution of Bbsl species are probably associated with the local environmental conditions and vertebrate host spectrum. Due to the presence of Borrelia species pathogenic to humans, all explored sites can be ranked as areas with high epidemiological risk.

Molecular characterisation of three Ixodes (Pholeoixodes) species (Ixodida, Ixodidae) and the first record of Ixodes (Pholeoixodes) kaiseri from Slovakia.

A study of ticks on wildlife was carried out in the area of Levice, Bratislava, Stupava, and Vrbovce (south-western Slovakia) during 2021 and 2022. Overall, 512 ticks were collected from 51 individuals of six wild mammalian species. Eight tick species were identified, namely Dermacentorreticulatus, D.marginatus, Haemaphysalisinermis, H.concinna, Ixodesricinus, I.hexagonus, and two Ixodes spp. Ixodeshexagonus were collected from northern white-breasted hedgehogs (Erinaceusroumanicus), females belonging to Ixodes spp. were collected from red fox (Vulpesvulpes) and nymphs from European badger (Melesmeles). Ixodeshexagonus and the Ixodes spp. were identified morphologically and molecularly based on sequences of fragments of two mitochondrial genes, COI and 16S rRNA. Molecular analysis of Ixodes spp. confirmed the identity of Ixodeskaiseri Arthur, 1957 and I.canisuga (Johnston, 1849). Sequence analyses show that the I.kaiseri isolate from Slovakia is identical to I.kaiseri isolates from Romania, Poland, Germany, Turkey, and Croatia. We demonstrate for the first time the presence of I.kaiseri in Slovakia using both morphological and molecular methods.

Discovery of the Role of Tick Salivary Glands in Enhancement of Virus Transmission-Beginning of an Exciting Story.

There is increasing evidence that arthropod-borne pathogens exploit saliva of their vectors during the transmission process to vertebrate hosts. Extensive research of the composition of tick saliva and its role in blood-feeding and transmission of pathogens started in the late 1980s and led to a number of discoveries on the composition and function of salivary molecules, some of which are associated with pathogen transmission. The study by Jones et al. published in 1989 can be ranked among the pioneer works in this field as it demonstrated for the first time the role of tick salivary glands in enhancement of transmission of a tick-borne virus. Thogoto virus was used in the model and subsequently similar results were obtained for tick-borne encephalitis virus. After a relatively silent period of almost 20 years, interest in tick-arbovirus-host interactions emerged again in the 2010s. However, no particular salivary molecule(s) enhancing virus transmission has (have) been identified to date. Intensive research in this field will certainly lead to new discoveries with future implications in the control of transmission of dangerous tick-borne viruses.

What do we know about the microbiome of I. ricinus?

I. ricinus is an obligate hematophagous parasitic arthropod that is responsible for the transmission of a wide range of zoonotic pathogens including spirochetes of the genus Borrelia, Rickettsia spp., C. burnetii, Anaplasma phagocytophilum and Francisella tularensis, which are part the tick´s microbiome. Most of the studies focus on "pathogens" and only very few elucidate the role of "non-pathogenic" symbiotic microorganisms in I. ricinus. While most of the members of the microbiome are leading an intracellular lifestyle, they are able to complement tick´s nutrition and stress response having a great impact on tick´s survival and transmission of pathogens. The composition of the tick´s microbiome is not consistent and can be tied to the environment, tick species, developmental stage, or specific organ or tissue. Ovarian tissue harbors a stable microbiome consisting mainly but not exclusively of endosymbiotic bacteria, while the microbiome of the digestive system is rather unstable, and together with salivary glands, is mostly comprised of pathogens. The most prevalent endosymbionts found in ticks are Rickettsia spp., Ricketsiella spp., Coxiella-like and Francisella-like endosymbionts, Spiroplasma spp. and Candidatus Midichloria spp. Since microorganisms can modify ticks' behavior, such as mobility, feeding or saliva production, which results in increased survival rates, we aimed to elucidate the potential, tight relationship, and interaction between bacteria of the I. ricinus microbiome. Here we show that endosymbionts including Coxiella-like spp., can provide I. ricinus with different types of vitamin B (B2, B6, B7, B9) essential for eukaryotic organisms. Furthermore, we hypothesize that survival of Wolbachia spp., or the bacterial pathogen A. phagocytophilum can be supported by the tick itself since coinfection with symbiotic Spiroplasma ixodetis provides I. ricinus with complete metabolic pathway of folate biosynthesis necessary for DNA synthesis and cell division. Manipulation of tick´s endosymbiotic microbiome could present a perspective way of I. ricinus control and regulation of spread of emerging bacterial pathogens.

Bartonella, Rickettsia, Babesia, and Hepatozoon Species in Fleas (Siphonaptera) Infesting Small Mammals of Slovakia (Central Europe).

Fleas (Siphonaptera) as obligate, blood-feeding ectoparasites are, together with ticks, hosted by small mammals and can transmit causative agents of serious infections. This study aimed to determine and characterize the presence and genetic diversity of Bartonella, Rickettsia, and apicomplexan parasites (Babesia, Hepatozoon) in fleas feeding on small mammals from three different habitat types (suburban, natural, and rural) in Slovakia. The most common pathogen in the examined fleas was Bartonella spp. (33.98%; 95% CI: 30.38-37.58), followed by Rickettsia spp. (19.1%; 95% CI: 16.25-22.24) and apicomplexan parasites (4.36%; 95% CI: 2.81-5.91). Bartonella strains belonging to B. taylorii, B. grahamii, B. elizabethae, Bartonella sp. wbs11, and B. rochalimae clades were identified in Ctenophthalmus agyrtes, C. congener, C. assimilis, C. sciurorum, C. solutus, C. bisoctodentatus, Palaeopsylla similis, Megabothris turbidus, and Nosopsyllus fasciatus within all habitats. The presence of Rickettsia helvetica, R. monacensis, and rickettsiae, belonging to the R. akari and R. felis clusters, and endosymbionts with a 96-100% identity with the Rickettsia endosymbiont of Nosopsyllus laeviceps laeviceps were also revealed in C. agyrtes, C. solutus, C. assimilis, C. congener, M. turbidus, and N. fasciatus. Babesia and Hepatozoon DNA was detected in the fleas from all habitat types. Hepatozoon sp. was detected in C. agyrtes, C. assimilis, and M. turbidus, while Babesia microti was identified from C. agyrtes, C. congener, and P. similis. The present study demonstrated the presence of zoonotic pathogens in fleas, parasitizing the wild-living small mammals of southwestern and central Slovakia and widens our knowledge of the ecology and genomic diversity of Bartonella, Rickettsia, Babesia, and Hepatozoon.

Ticks and their epidemiological role in Slovakia: from the past till present.

In Slovakia, 22 tick species have been found to occur to date. Among them, Ixodes ricinus, Dermacentor reticulatus, D. marginatus and marginally Haemaphysalis concinna, H. inermis and H. punctata have been identified as the species of public health relevance. Ticks in Slovakia were found to harbour and transmit zoonotic and/or potentially zoonotic agents such as tick-borne encephalitis virus (TBEV), spirochaetes of the Borrelia burgdorferi sensu lato (s.l.) complex, the relapsing fever sprirochaete Borrelia miyamotoi, bacteria belonging to the orders Rickettsiales (Rickettsia spp., Anaplasma phagocytophilum, Neoehrlichia mikurensis), Legionellales (Coxiella burnetii), and Thiotrichales (Francisella tularensis), and Babesia spp. parasites (order Piroplasmida). Ixodes ricinus is the principal vector of the largest variety of microorganisms including viruses, bacteria and piroplasms. TBEV, B. burgdorferi s.l., rickettsiae of the spotted fever group, C. burnetii and F. tularensis have been found to cause serious diseases in humans, whereas B. miyamotoi, A. phagocytophilum, N. mikurensis, Babesia microti, and B. venatorum pose lower or potential risk to humans. Distribution of TBEV has a focal character. During the last few decades, new tick-borne encephalitis (TBE) foci and their spread to new areas have been registered and TBE incidence rates have increased. Moreover, Slovakia reports the highest rates of alimentary TBE infections among the European countries. Lyme borreliosis (LB) spirochaetes are spread throughout the distribution range of I. ricinus. Incidence rates of LB have shown a slightly increasing trend since 2010. Only a few sporadic cases of human rickettsiosis, anaplasmosis and babesiosis have been confirmed thus far in Slovakia. The latest large outbreaks of Q fever and tularaemia were recorded in 1993 and 1967, respectively. Since then, a few human cases of Q fever have been reported almost each year. Changes in the epidemiological characteristics and clinical forms of tularaemia have been observed during the last few decades. Global changes and development of modern molecular tools led to the discovery and identification of emerging or new tick-borne microorganisms and symbionts with unknown zoonotic potential. In this review, we provide a historical overview of research on ticks and tick-borne pathogens in Slovakia with the most important milestones and recent findings, and outline future directions in the investigation of ticks as ectoparasites and vectors of zoonotic agents and in the study of tick-borne diseases.

Evolutionary Relationships of Ljungan Virus Variants Circulating in Multi-Host Systems across Europe.

The picornavirus named 'Ljungan virus' (LV, species Parechovirus B) has been detected in a dozen small mammal species from across Europe, but detailed information on its genetic diversity and host specificity is lacking. Here, we analyze the evolutionary relationships of LV variants circulating in free-living mammal populations by comparing the phylogenetics of the VP1 region (encoding the capsid protein and associated with LV serotype) and the 3Dpol region (encoding the RNA polymerase) from 24 LV RNA-positive animals and a fragment of the 5' untranslated region (UTR) sequence (used for defining strains) in sympatric small mammals. We define three new VP1 genotypes: two in bank voles (Myodes glareolus) (genotype 8 from Finland, Sweden, France, and Italy, and genotype 9 from France and Italy) and one in field voles (Microtus arvalis) (genotype 7 from Finland). There are several other indications that LV variants are host-specific, at least in parts of their range. Our results suggest that LV evolution is rapid, ongoing and affected by genetic drift, purifying selection, spillover and host evolutionary history. Although recent studies suggest that LV does not have zoonotic potential, its widespread geographical and host distribution in natural populations of well-characterized small mammals could make it useful as a model for studying RNA virus evolution and transmission.

Geographical Distribution and Genetic Diversity of Bank Vole Hepaciviruses in Europe.

The development of new diagnostic methods resulted in the discovery of novel hepaciviruses in wild populations of the bank vole (Myodes glareolus, syn. Clethrionomys glareolus). The naturally infected voles demonstrate signs of hepatitis similar to those induced by hepatitis C virus (HCV) in humans. The aim of the present research was to investigate the geographical distribution of bank vole-associated hepaciviruses (BvHVs) and their genetic diversity in Europe. Real-time reverse transcription polymerase chain reaction (RT-qPCR) screening revealed BvHV RNA in 442 out of 1838 (24.0%) bank voles from nine European countries and in one of seven northern red-backed voles (Myodes rutilus, syn. Clethrionomys rutilus). BvHV RNA was not found in any other small mammal species (n = 23) tested here. Phylogenetic and isolation-by-distance analyses confirmed the occurrence of both BvHV species (Hepacivirus F and Hepacivirus J) and their sympatric occurrence at several trapping sites in two countries. The broad geographical distribution of BvHVs across Europe was associated with their presence in bank voles of different evolutionary lineages. The extensive geographical distribution and high levels of genetic diversity of BvHVs, as well as the high population fluctuations of bank voles and occasional commensalism in some parts of Europe warrant future studies on the zoonotic potential of BvHVs.

Hymenopteran Parasitoids of Hard Ticks in Western Africa and the Russian Far East.

Some parasitoids of the genus Ixodiphagus (Hymenoptera, Chalcidoidea: Encyrtidae) are well-known natural enemies of ticks. In this study, we investigate the occurrence of parasitoid wasps in adult hard ticks from Western Africa (Côte d'Ivoire and Senegal) and Far Eastern Europe (Russia) using molecular methods. The morphological identification allowed the classification of 785 collected specimens of six species of ticks: Rhipicephalus (Boophilus) microplus (41%), Ixodes persulcatus (33%), Dermacentor silvarum (11%), Haemaphysalis concinna (7%), Amblyomma variegatum (5%), and Haemaphysalis japonica (3%). The newly developed MALDI-TOF MS protocol identified tick species in spite of their different storage (dried or in 70% ethanol) conditions for a long period. Molecular screening of ticks by a new standard PCR system developed in this study revealed the presence of parasitoid wasp DNA in 3% (28/785) of analyzed ticks. Ixodiphagus hookeri was detected in 86% (24/28) of infested ticks, including 13 I. persulcatus, 9 R (B) microplus, and one H. concinna and D. silvarum. While an unidentified parasitoid wasp species from the subfamily Aphidiinae and Braconidae family was detected in the remaining 14% (4/28) infested ticks. These infested ticks were identified as I. persulcatus. Our findings highlight the need for further studies to clarify the species diversity of parasitoid infesting ticks by combining molecular and morphological features. The novel molecular and MALDI-TOF MS protocols could be effective tools for the surveillance and characterization of these potential bio-control agents of ticks.

Ticks and the effects of their saliva on growth factors involved in skin wound healing.

Ticks are unique hematophagous arthropods and possess an astounding array of salivary molecules that ensure their unnoticed and prolonged attachment to the host skin. Furthermore, ticks are very effective vectors of a diverse spectrum of pathogens. In order to feed, tick chelicerae cut the host epidermis and their hypostome penetrates through the layers of the skin. As a result of laceration of the skin and rupturing blood vessels, a pool of blood is formed in the dermis, serving for intermittent blood sucking and secretion of saliva. Cutaneous injury caused by tick mouthparts should normally elicit wound healing, a complex biological process coordinated by interaction among different host cells, numerous signalling pathways and by a variety of soluble factors including growth factors. Growth factors, endogenous signalling proteins involved in various biological events, are key players in all phases of the skin repair process. Maintaining feeding site integrity by overcoming sequential phases of wound healing is particularly important for ixodid ticks and is governed by bioactive molecules in their saliva. Tick saliva is a complex mixture of proteins, peptides, and non-peptide molecules and its composition depends on the feeding phase, tick developmental stage, gender and/or the presence/absence of microbial agents. In addition to already demonstrated anti-haemostatic, anti-cytokine and anti-chemokine activities, anti-growth factors activities were also detected in saliva of some tick species. In consequence of counteracting host defences by ticks, tick-borne pathogens can be transmitted to and disseminated in the host. Elucidation of the complex interplay between ticks - pathogens - host cutaneous immunity could lead to improved vector and pathogens control strategies. Additionally, tick saliva bioactive molecules have a promising therapeutic perspective to cure some human diseases associated with dysregulation of specific cytokines/growth factors and alterations in their signalling pathways.

Isolation and partial characterisation of a novel Trypanosoma from the tick Ixodes ricinus.

Trypanosomes have long been recognised as being amongst the most important protozoan parasites of vertebrates, from both medical and veterinary perspectives. Whilst numerous insect species have been identified as vectors, the role of ticks is less well understood. Here we report the isolation and partial molecular characterisation of a novel trypanosome from questing Ixodes ricinus ticks collected in Slovakia. The trypanosome was isolated in tick cell culture and then partially characterised by microscopy and amplification of fragments of the 18S rRNA and 24Sα rDNA genes. Analysis of the resultant sequences suggests that the trypanosome designated as Trypanosoma sp. Bratislava1 may be a new species closely related to several species or strains of trypanosomes isolated from, or detected in, ticks in South America and Asia, and to Trypanosoma caninum isolated from dogs in Brazil. This study highlights the potential involvement of ixodid ticks in the epidemiology of trypanosomes, as well as the use of tick cell lines for isolation of such tick-borne protozoa. Further studies are required to investigate the epidemiology, transmission and life cycle of this putative novel species.

Geographical Distribution of Ljungan Virus in Small Mammals in Europe.

Ljungan virus (LV), which belongs to the Parechovirus genus in the Picornaviridae family, was first isolated from bank voles (Myodes glareolus) in Sweden in 1998 and proposed as a zoonotic agent. To improve knowledge of the host association and geographical distribution of LV, tissues from 1685 animals belonging to multiple rodent and insectivore species from 12 European countries were screened for LV-RNA using reverse transcriptase (RT)-PCR. In addition, we investigated how the prevalence of LV-RNA in bank voles is associated with various intrinsic and extrinsic factors. We show that LV is widespread geographically, having been detected in at least one host species in nine European countries. Twelve out of 21 species screened were LV-RNA PCR positive, including, for the first time, the red vole (Myodes rutilus) and the root or tundra vole (Alexandromys formerly Microtus oeconomus), as well as in insectivores, including the bicolored white-toothed shrew (Crocidura leucodon) and the Valais shrew (Sorex antinorii). Results indicated that bank voles are the main rodent host for this virus (overall RT-PCR prevalence: 15.2%). Linear modeling of intrinsic and extrinsic factors that could impact LV prevalence showed a concave-down relationship between body mass and LV occurrence, so that subadults had the highest LV positivity, but LV in older animals was less prevalent. Also, LV prevalence was higher in autumn and lower in spring, and the amount of precipitation recorded during the 6 months preceding the trapping date was negatively correlated with the presence of the virus. Phylogenetic analysis on the 185 base pair species-specific sequence of the 5' untranslated region identified high genetic diversity (46.5%) between 80 haplotypes, although no geographical or host-specific patterns of diversity were detected.

Tick-Borne Encephalitis Virus Infection Alters the Sialome of Ixodes ricinus Ticks During the Earliest Stages of Feeding.

Ticks are hematophagous arthropods that transmit a number of pathogens while feeding. Among these is tick-borne encephalitis virus (TBEV), a flavivirus transmitted by Ixodes ricinus ticks in the temperate zone of Europe. The infection results in febrile illness progressing to encephalitis and meningitis with a possibility of fatality or long-term neurological sequelae. The composition of tick saliva plays an essential role in the initial virus transmission during tick feeding. Ticks secrete a diverse range of salivary proteins to modulate the host response, such as lipocalins to control the itch and inflammatory response, and both proteases and protease inhibitors to prevent blood coagulation. Here, the effect of viral infection of adult females of Ixodes ricinus was studied with the goal of determining how the virus alters the tick sialome to modulate host tissue response at the site of infection. Uninfected ticks or those infected with TBEV were fed on mice and removed and dissected one- and 3-h post-attachment. RNA from the salivary glands of these ticks, as well as from unfed ticks, was extracted and subjected to next-generation sequencing to determine the expression of key secreted proteins at each timepoint. Genes showing statistically significant up- or down-regulation between infected and control ticks were selected and compared to published literature to ascertain their function. From this, the effect of tick viral infection on the modulation of the tick-host interface was determined. Infected ticks were found to differentially express a number of uncategorized genes, proteases, Kunitz-type serine protease inhibitors, cytotoxins, and lipocalins at different timepoints. These virus-induced changes to the tick sialome may play a significant role in facilitating virus transmission during the early stages of tick feeding.

Presence of Roe Deer Affects the Occurrence of Anaplasma phagocytophilum Ecotypes in Questing Ixodes ricinus in Different Habitat Types of Central Europe.

The way in which European genetic variants of Anaplasma phagocytophilum circulate in their natural foci and which variants cause disease in humans or livestock remains thus far unclear. Red deer and roe deer are suggested to be reservoirs for some European A. phagocytophilum strains, and Ixodes ricinus is their principal vector. Based on groEL gene sequences, five A. phagocytophilum ecotypes have been identified. Ecotype I is associated with the broadest host range, including strains that cause disease in domestic animals and humans. Ecotype II is associated with roe deer and does not include zoonotic strains. In the present study, questing I. ricinus were collected in urban, pasture, and natural habitats in the Czech Republic, Germany, and Slovakia. A fragment of the msp2 gene of A. phagocytophilum was amplified by real-time PCR in DNA isolated from ticks. Positive samples were further analyzed by nested PCRs targeting fragments of the 16S rRNA and groEL genes, followed by sequencing. Samples were stratified according to the presence/absence of roe deer at the sampling sites. Geographic origin, habitat, and tick stage were also considered. The probability that A. phagocytophilum is a particular ecotype was estimated by a generalized linear model. Anaplasma phagocytophilum was identified by genetic typing in 274 I. ricinus ticks. The majority belonged to ecotype I (63.9%), 28.5% were ecotype II, and both ecotypes were identified in 7.7% of ticks. Ecotype II was more frequently identified in ticks originating from a site with presence of roe deer, whereas ecotype I was more frequent in adult ticks than in nymphs. Models taking into account the country-specific, site-specific, and habitat-specific aspects did not improve the goodness of the fit. Thus, roe deer presence in a certain site and the tick developmental stage are suggested to be the two factors consistently influencing the occurrence of a particular A. phagocytophilum ecotype in a positive I. ricinus tick.

Deciphering Biological Processes at the Tick-Host Interface Opens New Strategies for Treatment of Human Diseases.

Ticks are obligatory blood-feeding ectoparasites, causing blood loss and skin damage in their hosts. In addition, ticks also transmit a number of various pathogenic microorganisms that cause serious diseases in humans and animals. Ticks evolved a wide array of salivary bioactive compounds that, upon injection into the host skin, inhibit or modulate host reactions such as hemostasis, inflammation and wound healing. Modulation of the tick attachment site in the host skin involves mainly molecules which affect physiological processes orchestrated by cytokines, chemokines and growth factors. Suppressing host defense reactions is crucial for tick survival and reproduction. Furthermore, pharmacologically active compounds in tick saliva have a promising therapeutic potential for treatment of some human diseases connected with disorders in hemostasis and immune system. These disorders are often associated to alterations in signaling pathways and dysregulation or overexpression of specific cytokines which, in turn, affect mechanisms of angiogenesis, cell motility and cytoskeletal regulation. Moreover, tick salivary molecules were found to exert cytotoxic and cytolytic effects on various tumor cells and have anti-angiogenic properties. Elucidation of the mode of action of tick bioactive molecules on the regulation of cell processes in their mammalian hosts could provide new tools for understanding the complex changes leading to immune disorders and cancer. Tick bioactive molecules may also be exploited as new pharmacological inhibitors of the signaling pathways of cytokines and thus help alleviate patient discomfort and increase patient survival. We review the current knowledge about tick salivary peptides and proteins that have been identified and functionally characterized in in vitro and/or in vivo models and their therapeutic perspective.

Anaplasma phagocytophilum evolves in geographical and biotic niches of vertebrates and ticks.

BACKGROUND: Anaplasma phagocytophilum is currently regarded as a single species. However, molecular studies indicate that it can be subdivided into ecotypes, each with distinct but overlapping transmission cycle. Here, we evaluate the interactions between and within clusters of haplotypes of the bacterium isolated from vertebrates and ticks, using phylogenetic and network-based methods. METHODS: The presence of A. phagocytophilum DNA was determined in ticks and vertebrate tissue samples. A fragment of the groEl gene was amplified and sequenced from qPCR-positive lysates. Additional groEl sequences from ticks and vertebrate reservoirs were obtained from GenBank and through literature searches, resulting in a dataset consisting of 1623 A. phagocytophilum field isolates. Phylogenetic analyses were used to infer clusters of haplotypes and to assess phylogenetic clustering of A. phagocytophilum in vertebrates or ticks. Network-based methods were used to resolve host-vector interactions and their relative importance in the segregating communities of haplotypes. RESULTS: Phylogenetic analyses resulted in 199 haplotypes within eight network-derived clusters, which were allocated to four ecotypes. The interactions of haplotypes between ticks, vertebrates and geographical origin, were visualized and quantified from networks. A high number of haplotypes were recorded in the tick Ixodes ricinus. Communities of A. phagocytophilum recorded from Korea, Japan, Far Eastern Russia, as well as those associated with rodents had no links with the larger set of isolates associated with I. ricinus, suggesting different evolutionary pressures. Rodents appeared to have a range of haplotypes associated with either Ixodes trianguliceps or Ixodes persulcatus and Ixodes pavlovskyi. Haplotypes found in rodents in Russia had low similarities with those recorded in rodents in other regions and shaped separate communities. CONCLUSIONS: The groEl gene fragment of A. phagocytophilum provides information about spatial segregation and associations of haplotypes to particular vector-host interactions. Further research is needed to understand the circulation of this bacterium in the gap between Europe and Asia before the overview of the speciation features of this bacterium is complete. Environmental traits may also play a role in the evolution of A. phagocytophilum in ecotypes through yet unknown relationships.

Diverse tick-borne microorganisms identified in free-living ungulates in Slovakia.

BACKGROUND: Free-living ungulates are hosts of ixodid ticks and reservoirs of tick-borne microorganisms in central Europe and many regions around the world. Tissue samples and engorged ticks were obtained from roe deer, red deer, fallow deer, mouflon, and wild boar hunted in deciduous forests of south-western Slovakia. DNA isolated from these samples was screened for the presence of tick-borne microorganisms by PCR-based methods. RESULTS: Ticks were found to infest all examined ungulate species. The principal infesting tick was Ixodes ricinus, identified on 90.4% of wildlife, and included all developmental stages. Larvae and nymphs of Haemaphysalis concinna were feeding on 9.6% of wildlife. Two specimens of Dermacentor reticulatus were also identified. Ungulates were positive for A. phagocytophilum and Theileria spp. Anaplasma phagocytophilum was found to infect 96.1% of cervids, 88.9% of mouflon, and 28.2% of wild boar, whereas Theileria spp. was detected only in cervids (94.6%). Importantly, a high rate of cervids (89%) showed mixed infections with both these microorganisms. In addition to A. phagocytophilum and Theileria spp., Rickettsia helvetica, R. monacensis, unidentified Rickettsia sp., Coxiella burnetii, "Candidatus Neoehrlichia mikurensis", Borrelia burgdorferi (s.l.) and Babesia venatorum were identified in engorged I. ricinus. Furthermore, A. phagocytophilum, Babesia spp. and Theileria spp. were detected in engorged H. concinna. Analysis of 16S rRNA and groEL gene sequences revealed the presence of five and two A. phagocytophilum variants, respectively, among which sequences identified in wild boar showed identity to the sequence of the causative agent of human granulocytic anaplasmosis (HGA). Phylogenetic analysis of Theileria 18S rRNA gene sequences amplified from cervids and engorged I. ricinus ticks segregated jointly with sequences of T. capreoli isolates into a moderately supported monophyletic clade. CONCLUSIONS: The findings indicate that free-living ungulates are reservoirs for A. phagocytophilum and Theileria spp. and engorged ixodid ticks attached to ungulates are good sentinels for the presence of agents of public and veterinary concern. Further analyses of the A. phagocytophilum genetic variants and Theileria species and their associations with vector ticks and free-living ungulates are required.

Effect of Climate and Land Use on the Spatio-Temporal Variability of Tick-Borne Bacteria in Europe.

The incidence of tick-borne diseases caused by Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum and Rickettsia spp. has been rising in Europe in recent decades. Early pre-assessment of acarological hazard still represents a complex challenge. The aim of this study was to model Ixodes ricinus questing nymph density and its infection rate with B. burgdorferi s.l., A. phagocytophilum and Rickettsia spp. in five European countries (Italy, Germany, Czech Republic, Slovakia, Hungary) in various land cover types differing in use and anthropisation (agricultural, urban and natural) with climatic and environmental factors (Normalized Difference Vegetation Index (NDVI), Normalized Difference Water Index (NDWI), Land Surface Temperature (LST) and precipitation). We show that the relative abundance of questing nymphs was significantly associated with climatic conditions, such as higher values of NDVI recorded in the sampling period, while no differences were observed among land use categories. However, the density of infected nymphs (DIN) also depended on the pathogen considered and land use. These results contribute to a better understanding of the variation in acarological hazard for Ixodes ricinus transmitted pathogens in Central Europe and provide the basis for more focused ecological studies aimed at assessing the effect of land use in different sites on tick-host pathogens interaction.