Identification and expression of short neuropeptide F and its receptors in the tick Ixodes ricinus.

In Europe, the tick Ixodes ricinus is the most important vector of numerous pathogens that are transmitted during blood feeding on their vertebrate hosts. To elucidate mechanisms controlling blood intake and associated transmission of pathogens we identified and described expression of short neuropeptide F (sNPF) and its receptors which are known to regulate feeding in insects. Using in situ hybridization (ISH) and immunohistochemistry (IHC) we stained numerous neurons producing sNPF in the central nervous system (CNS; synganglion), while a few peripheral neurons were detected anteriorly to the synganglion, and on the surface of the hindgut and leg muscles. Apparent sNPF expression was also found in enteroendocrine cells individually scattered in anterior lobes of the midgut. In silico analyses and BLAST search for sNPF receptors revealed two putative G protein-coupled receptors (sNPFR1 and sNPFR2) in the I. ricinus genome. Aequorin-based functional assay in CHO cells showed that both receptors were specific and sensitive to sNPF in nanomolar concentrations. Increased expression levels of these receptors in the gut during blood intake suggest that sNPF signaling may be involved in regulation of feeding and digestion processes of I. ricinus.

Dermacentor reticulatus is a vector of tick-borne encephalitis virus.

Tick-borne encephalitis virus (TBEV; family Flaviviridae) is the most medically important tick-borne virus in Europe and Asia. Ixodes ricinus and I. persulcatus ticks are considered to be the main vector ticks of TBEV in nature due to their specific ecological associations with the vertebrate hosts. Nevertheless, recent TBEV prevalence studies in ticks suggest that Dermacentor reticulatus ticks might play a relevant role in the maintenance of TBEV in nature. The goal of this study was to evaluate the vector competency of D. reticulatus for TBEV through experimental tick infections and comparative in vivo transmission studies involving D. reticulatus and I. ricinus ticks. We observed that after a transcoxal micro-capillary inoculation, adult female D. reticulatus ticks efficiently replicated TBEV during the observed period of 21 days. The mean virus load reached up to 2.5 × 105 gene copies and 6.4 × 104 plaque forming units per tick. The infected D. reticulatus ticks were able to transmit the virus to mice. The course of infection in mice was comparable to the infection after a tick bite by I. ricinus while the virus spread and clearance was slightly faster. Moreover, D. reticulatus ticks were capable of tick-to-tick non-viraemic transmission of TBEV to the Haemaphysalis inermis nymphs during co-feeding on the same animal. The co-feeding transmission efficiency was overall slightly lower (up to 54 %) in comparison with I. ricinus (up to 94 %) and peaked 1 day later, at day 3. In conclusion, our study demonstrated that D. reticulatus is a biologically effective vector of TBEV. In line with the recent reports of its high TBEV prevalence in nature, our data indicate that in some endemic foci, D. reticulatus might be an underrecognized TBEV vector which contributes to the expansion of the TBEV endemic areas.

A Versatile Model of Hard Tick Infestation on Laboratory Rabbits.

The use of live animals in tick research is crucial for a variety of experimental purposes including the maintenance of hard tick colonies in the laboratory. In ticks, all developmental stages (except egg) are hematophagous, and acquiring a blood-meal when attached to their vertebrate hosts is essential for the successful completion of their life cycle. Here we demonstrate a simple method that uses easily openable capsules for feeding of hard ticks on rabbits. The advantages of the proposed method include its simplicity, short duration and most importantly versatile adjustment to the needs of specific experimental requirements. The method makes possible the use of multiple chambers (of various sizes) on the same animal, which permits feeding of multiple stages or different experimental groups while reducing the overall animal requirement. The non-irritating and easily accessible materials used minimizes discomfort to the animals, which can be easily recovered from an experiment and offered for adoption or reused if the ethical protocol allows it.

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.

Transcriptional Immunoprofiling at the Tick-Virus-Host Interface during Early Stages of Tick-Borne Encephalitis Virus Transmission.

Emerging and re-emerging diseases transmitted by blood feeding arthropods are significant global public health problems. Ticks transmit the greatest variety of pathogenic microorganisms of any blood feeding arthropod. Infectious agents transmitted by ticks are delivered to the vertebrate host together with saliva at the bite site. Tick salivary glands produce complex cocktails of bioactive molecules that facilitate blood feeding and pathogen transmission by modulating host hemostasis, pain/itch responses, wound healing, and both innate and adaptive immunity. In this study, we utilized Illumina Next Generation Sequencing to characterize the transcriptional immunoprofile of cutaneous immune responses to Ixodes ricinus transmitted tick-borne encephalitis virus (TBEV). A comparative immune gene expression analysis of TBEV-infected and uninfected tick feeding sites was performed. Our analysis reveals that ticks create an inflammatory environment at the bite site during the first 3 h of feeding, and significant differences in host responses were observed between TBEV-infected and uninfected tick feeding. Gene-expression analysis reveals modulation of inflammatory genes after 1 and 3 h of TBEV-infected tick feeding. Transcriptional levels of genes specific to chemokines and cytokines indicated a neutrophil-dominated immune response. Immunohistochemistry of the tick feeding site revealed that mononuclear phagocytes and fibroblasts are the primary target cells for TBEV infection and did not detect TBEV antigens in neutrophils. Together, the transcriptional and immunohistochemistry results suggest that early cutaneous host responses to TBEV-infected tick feeding are more inflammatory than expected and highlight the importance of inflammatory chemokine and cytokine pathways in tick-borne flavivirus transmission.

Tick-Borne Transmission of Murine Gammaherpesvirus 68.

Herpesviruses are a large group of DNA viruses infecting mainly vertebrates. Murine gammaherpesvirus 68 (MHV68) is often used as a model in studies of the pathogenesis of clinically important human gammaherpesviruses such as Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus. This rodent virus appears to be geographically widespread; however, its natural transmission cycle is unknown. Following detection of MHV68 in field-collected ticks, including isolation of the virus from tick salivary glands and ovaries, we investigated whether MHV68 is a tick-borne virus. Uninfected Ixodes ricinus ticks were shown to acquire the virus by feeding on experimentally infected laboratory mice. The virus survived tick molting, and the molted ticks transmitted the virus to uninfected laboratory mice on which they subsequently fed. MHV68 was isolated from the tick salivary glands, consistent with transmission via tick saliva. The virus survived in ticks without loss of infectivity for at least 120 days, and subsequently was transmitted vertically from one tick generation to the next, surviving more than 500 days. Furthermore, the F1 generation (derived from F0 infected females) transmitted MHV68 to uninfected mice on which they fed, with MHV68 M3 gene transcripts detected in blood, lung, and spleen tissue of mice on which F1 nymphs and F1 adults engorged. These experimental data fulfill the transmission criteria that define an arthropod-borne virus (arbovirus), the largest biological group of viruses. Currently, African swine fever virus (ASFV) is the only DNA virus recognized as an arbovirus. Like ASFV, MHV68 showed evidence of pathogenesis in ticks. Previous studies have reported MHV68 in free-living ticks and in mammals commonly infested with I. ricinus, and neutralizing antibodies to MHV68 have been detected in large mammals (e.g., deer) including humans. Further studies are needed to determine if these reports are the result of tick-borne transmission of MHV68 in nature, and whether humans are at risk of infection.

Diversity and prevalence of Bartonella species in small mammals from Slovakia, Central Europe.

Wild-living rodents are important hosts for zoonotic pathogens. Bartonella infections are widespread in rodents; however, in Slovakia, knowledge on the prevalence of these bacteria in small mammals is limited. We investigated the prevalence and diversity of Bartonella species in the spleens of 640 rodents of six species (Apodemus flavicollis, Apodemus sylvaticus, Myodes glareolus, Microtus arvalis, Microtus subterraneus, and Micromys minutus) and in the European mole (Talpa europaea) from three different habitat types in south-western and central Slovakia. Overall, the prevalence of Bartonella spp. in rodents was 64.8%; a rate of 73.8% was found in natural habitat (deciduous forest), 56.0% in suburban forest park and 64.9% in rural habitat. Bartonella spp. were detected in 63.0% of A. flavicollis, 69% of My. glareolus and 61.1% of M. arvalis and in T. europaea. However, Bartonella were not found in the other examined rodents. Molecular analyses of the 16S-23S rRNA intergenic spacer region revealed the presence of four different Bartonella spp. clusters. We identified B. taylorii, B. rochalimae, B. elizabethae, B. grahamii and Bartonella sp. wbs11 in A. flavicollis and My. glareolus. Bartonella genotypes ascribed to B. taylorii and B. rochalimae were found in M. arvalis. B. taylorii was identified in T. europaea. Questing Ixodes ricinus ticks that were collected at the study sites were not infected with Bartonella. This study improves our understanding of the ecoepidemiology of Bartonella spp. in Europe and underlines the necessity for further research on Bartonella-host-vector associations and their consequences on animal and human health in Slovakia.

Molecular evidence of Rickettsia spp. in ixodid ticks and rodents in suburban, natural and rural habitats in Slovakia.

BACKGROUND: Natural foci of tick-borne spotted fever group (SFG) rickettsiae of public health concern have been found in Slovakia, but the role of rodents in their circulation is unclear. Ticks (Ixodes ricinus, Ixodes trianguliceps, Dermacentor marginatus, Dermacentor reticulatus, Haemaphysalis concinna and Haemaphysalis inermis) and tissues of rodents (Apodemus flavicollis, Apodemus sylvaticus, Myodes glareolus, Microtus arvalis, Microtus subterraneus and Micromys minutus) were examined for the presence of SFG rickettsiae and Coxiella burnetii by molecular methods. Suburban, natural and rural habitats were monitored to acquire information on the role of ticks and rodents in the agents' maintenance in various habitat types of Slovakia. RESULTS: The overall prevalence of rickettsial infection in questing I. ricinus and D. marginatus was 6.6% and 21.4%, respectively. Rickettsia helvetica, R. monacensis and non-identified rickettsial species were detected in I. ricinus, whereas R. slovaca and R. raoultii were identified in D. marginatus. Rickettsia spp.-infected I. ricinus occurred during the whole tick questing period. Rickettsia helvetica dominated (80.5%) followed by R. monacensis (6.5%). The species were present in all studied habitats. Rickettsia slovaca (66.7%) and R. raoultii (33.3%) were identified in D. marginatus from the rural habitat. Apodemus flavicollis was the most infested rodent species with I. ricinus, but My. glareolus carried the highest proportion of Rickettsia-positive I. ricinus larvae. Only 0.5% of rodents (A. flavicollis) and 5.2% of engorged I. ricinus removed from My. glareolus, A. flavicollis and M. arvalis were R. helvetica- and R. monacensis-positive. Coxiella burnetii was not detected in any of the tested samples. We hypothesize that rodents could play a role as carriers of infected ticks and contribute to the maintenance of rickettsial pathogens in natural foci. CONCLUSIONS: Long-term presence of SFG Rickettsia spp. was confirmed in questing ticks from different habitat types of Slovakia. The results suggest a human risk for infection with the pathogenic R. helvetica, R. monacensis, R. slovaca and R. raoultii.

An insight into the sialome of Hyalomma excavatum.

Tick saliva contains hundreds or thousands of proteins that help blood feeding by impairing their hosts' hemostasis, inflammation and immunity. Salivary gland transcriptomes allow the disclosure of this pharmacologically active potion that consists of several multi-gene families, many of which are tick-specific. We here report the "de novo" assembly of ∼138 million reads deriving from a cDNA library from salivary glands of adult male and female Hyalomma excavatum leading to the public deposition of 5337 coding sequences to GenBank. Among the deducted putative secreted proteins, metalloproteases, glycine rich proteins, mucins, anticoagulants of the madanin family and lipocalins were the most expressed. Novel protein families were identified. These sequences will permit proteomic studies aiming at identification of target antigens, epidemiological markers or salivary pharmaceuticals of interest, and contribute to our understanding of the fast evolution of the tick sialome.

Molecular detection and phylogenetic analysis of Hepatozoon spp. in questing Ixodes ricinus ticks and rodents from Slovakia and Czech Republic.

By amplification and sequencing of 18S rRNA gene fragments, Hepatozoon spp. DNA was detected in 0.08 % (4/5057) and 0.04 % (1/2473) of questing Ixodes ricinus ticks from Slovakia and Czech Republic, respectively. Hepatozoon spp. DNA was also detected in spleen and/or lungs of 4.45 % (27/606) of rodents from Slovakia. Prevalence of infection was significantly higher in Myodes glareolus (11.45 %) than in Apodemus spp. (0.28 %) (P < 0.001). Sequencing of 18S rRNA Hepatozoon spp. gene amplicons from I. ricinus showed 100 % identity with Hepatozoon canis isolates from red foxes or dogs in Europe. Phylogenetic analysis showed that at least two H. canis 18S rRNA genotypes exist in Slovakia of which one was identified also in the Czech Republic. The finding of H. canis in questing I. ricinus suggests the geographical spread of the parasite and a potential role of other ticks as its vectors in areas where Rhipicephalus sanguineus is not endemic. Sequencing of 18S rRNA gene amplicons from M. glareolus revealed the presence of two closely related genetic variants, Hepatozoon sp. SK1 and Hepatozoon sp. SK2, showing 99-100 % identity with isolates from M. glareolus from other European countries. Phylogenetic analysis demonstrates that 18S rRNA variants SK1 and SK2 correspond to previously described genotypes UR1 and UR2 of H. erhardovae, respectively. The isolate from Apodemus flavicollis (Hepatozoon sp. SK3b) was 99 % identical with isolates from reptiles in Africa and Asia. Further studies are necessary to identify the taxonomic status of Hepatozoon spp. parasitizing rodents in Europe and the host-parasite interactions in natural foci.

Tick-Borne Encephalitis Virus Structural Proteins Are the Primary Viral Determinants of Non-Viraemic Transmission between Ticks whereas Non-Structural Proteins Affect Cytotoxicity.

Over 50 million humans live in areas of potential exposure to tick-borne encephalitis virus (TBEV). The disease exhibits an estimated 16,000 cases recorded annually over 30 European and Asian countries. Conventionally, TBEV transmission to Ixodes spp. ticks occurs whilst feeding on viraemic animals. However, an alternative mechanism of non-viraemic transmission (NVT) between infected and uninfected ticks co-feeding on the same transmission-competent host, has also been demonstrated. Here, using laboratory-bred I. ricinus ticks, we demonstrate low and high efficiency NVT for TBEV strains Vasilchenko (Vs) and Hypr, respectively. These virus strains share high sequence similarity but are classified as two TBEV subtypes. The Vs strain is a Siberian subtype, naturally associated with I. persulcatus ticks whilst the Hypr strain is a European subtype, transmitted by I. ricinus ticks. In mammalian cell culture (porcine kidney cell line PS), Vs and Hypr induce low and high cytopathic effects (cpe), respectively. Using reverse genetics, we engineered a range of viable Vs/Hypr chimaeric strains, with substituted genes. No significant differences in replication rate were detected between wild-type and chimaeric viruses in cell culture. However, the chimaeric strain Vs[Hypr str] (Hypr structural and Vs non-structural genomic regions) demonstrated high efficiency NVT in I. ricinus whereas the counterpart Hypr[Vs str] was not transmitted by NVT, indicating that the virion structural proteins largely determine TBEV NVT transmission efficiency between ticks. In contrast, in cell culture, the extent of cpe was largely determined by the non-structural region of the TBEV genome. Chimaeras with Hypr non-structural genes were more cytotoxic for PS cells when compared with Vs genome-based chimaeras.

Babesia spp. in ticks and wildlife in different habitat types of Slovakia.

BACKGROUND: Babesiosis is an emerging and potentially zoonotic disease caused by tick-borne piroplasmids of the Babesia genus. New genetic variants of piroplasmids with unknown associations to vectors and hosts are recognized. Data on the occurrence of Babesia spp. in ticks and wildlife widen the knowledge on the geographical distribution and circulation of piroplasmids in natural foci. Questing and rodent-attached ticks, rodents, and birds were screened for the presence of Babesia-specific DNA using molecular methods. Spatial and temporal differences of Babesia spp. prevalence in ticks and rodents from two contrasting habitats of Slovakia with sympatric occurrence of Ixodes ricinus and Haemaphysalis concinna ticks and co-infections of Candidatus N. mikurensis and Anaplasma phagocytophilum were investigated. RESULTS: Babesia spp. were detected in 1.5 % and 6.6 % of questing I. ricinus and H. concinna, respectively. Prevalence of Babesia-infected I. ricinus was higher in a natural than an urban/suburban habitat. Phylogenetic analysis showed that Babesia spp. from I. ricinus clustered with Babesia microti, Babesia venatorum, Babesia canis, Babesia capreoli/Babesia divergens, and Babesia odocoilei. Babesia spp. amplified from H. concinna segregated into two monophyletic clades, designated Babesia sp. 1 (Eurasia) and Babesia sp. 2 (Eurasia), each of which represents a yet undescribed novel species. The prevalence of infection in rodents (with Apodemus flavicollis and Myodes glareolus prevailing) with B. microti was 1.3 % in an urban/suburban and 4.2 % in a natural habitat. The majority of infected rodents (81.3 %) were positive for spleen and blood and the remaining for lungs and/or skin. Rodent-attached I. ricinus (accounting for 96.3 %) and H. concinna were infected with B. microti, B. venatorum, B. capreoli/B. divergens, Babesia sp. 1 (Eurasia), and Babesia sp. 2 (Eurasia). All B. microti and B. venatorum isolates were identical to known zoonotic strains from Europe. Less than 1.0 % of Babesia-positive ticks and rodents carried Candidatus N. mikurensis or A. phagocytophilum. CONCLUSION: Our findings suggest that I. ricinus and rodents play important roles in the epidemiology of zoonotic Babesia spp. in south-western Slovakia. Associations with vertebrate hosts and the pathogenicity of Babesia spp. infecting H. concinna ticks need to be further explored.

Relative density of host-seeking ticks in different habitat types of south-western Slovakia.

Ixodes ricinus is a vector of microbial pathogens of medical and veterinary importance in Europe. Recently, increasing abundance of ticks has been observed in urban and suburban areas. The aim of this study was to investigate the tick species composition and examine correlations between local environmental variables and the relative density of host-seeking I. ricinus in two habitat types. Questing ticks were collected along six 100 m(2) transects in urban/suburban locations of Bratislava town, and in a non-fragmented deciduous forest in the Small Carpathians Mountains (south-western Slovakia) during 2011-2013. In total, 6015 I. ricinus were collected (3435 and 2580 in the urban/suburban and natural habitat, respectively), out of which over 80 % were nymphs. Haemaphysalis concinna comprised 1.3 % of the tick collections. Peak I. ricinus nymph and adult host-seeking activities were registered in April-June. Spatial and temporal variation in tick relative density and differences in the subadult/adult ratio were observed between habitats and between locations within the same habitat type. The relative density of questing I. ricinus nymphs correlated negatively with altitude, geographical aspect and saturation deficit in a 64-day period comprising the 8-day period including the date of tick sampling and previous 56 days. No significant correlation was found between roe deer density and questing nymph density. The study revealed the presence of abundant I. ricinus populations in green areas of Bratislava, suggesting a risk of exposure of town dwellers and domestic and companion animals to potentially infected ticks.

Candidatus Neoehrlichia mikurensis in ticks and rodents from urban and natural habitats of South-Western Slovakia.

BACKGROUND: Candidatus Neoehrlichia mikurensis (CNM) is an emerging tick-borne pathogen causing severe disease in immunocompromised patients. In Europe, Ixodes ricinus is the primary vector and rodents act as reservoir hosts. New data on the prevalence of CNM in ticks and rodents contribute to the knowledge on the distribution of endemic areas and circulation of the bacterium in natural foci. METHODS: Questing ticks were collected and rodents were trapped in urban/suburban and natural habitats in South-Western Slovakia from 2011 to 2014. DNA from questing and rodent-attached ticks and rodent tissues were screened for CNM by real-time PCR. Rodent spleen samples positive for CNM were characterised at the groEL gene locus. Spatial and temporal differences in CNM prevalence in ticks and rodents and co-infections of ticks with CNM and Anaplasma phagocytophilum were analysed. RESULTS: The presence of CNM was confirmed in questing and rodent-attached I. ricinus ticks and in rodents. Total prevalence in both ticks and rodents was significantly higher in the natural habitat (2.3% and 10.1%, respectively) than in the urban/suburban habitat (1.0% and 3.3%, respectively). No seasonal pattern in CNM prevalence in ticks was observed, but prevalence in rodents was higher in autumn than in spring. CNM was detected in Apodemus flavicollis, Myodes glareolus, Microtus arvalis and Micromys minutus, with the highest prevalence in M. arvalis (30%). By screening CNM dissemination in rodent tissues, infection was detected in lungs of all specimens with positive spleens and in blood, kidney, liver and skin of part of those individuals. Infection with CNM was detected in 1.3% of rodent attached I. ricinus ticks. Sequences of a fragment of the groEL gene from CNM-positive rodents showed a high degree of identity with sequences of the gene amplified from ticks and infected human blood from Europe. Only 0.1% of CNM-positive questing ticks carried A. phagocytophilum. Ticks infected with CNM prevailed in the natural habitat (67.2%), whereas ticks infected with A. phagocytophilum prevailed in the urban/suburban habitat (75.0%). CONCLUSION: The study confirmed the circulation of CNM between I. ricinus ticks and rodents in South-Western Slovakia, and indicates a potential risk of contracting human infections.

Anaplasma phagocytophilum prevalence in ticks and rodents in an urban and natural habitat in South-Western Slovakia.

BACKGROUND: Ixodes ricinus is the principal vector of Anaplasma phagocytophilum, the ethiological agent of granulocytic anaplasmosis in Europe. Anaplasmosis is an emerging zoonotic disease with a natural enzootic cycle. The reservoir competence of rodents is unclear. Monitoring of A. phagocytophilum prevalence in I. ricinus and rodents in various habitat types of Slovakia may contribute to the knowledge about the epidemiology of anaplasmosis in Central Europe. METHODS: Over 4400 questing ixodid ticks, 1000 rodent-attached ticks and tissue samples of 606 rodents were screened for A. phagocytophilum DNA by real-time PCR targeting the msp2 gene. Ticks and rodents were captured along six transects in an urban/suburban and natural habitat in south-western Slovakia during 2011-2014. Estimates of wildlife (roe deer, red deer, fallow deer, mouflon, wild boar) densities in the study area were taken from hunter's yearly reports. Spatial and temporal differences in A. phagocytophilum prevalence in questing I. ricinus and relationships with relative abundance of ticks and wildlife were analysed. RESULTS: Overall prevalence of A. phagocytophilum in questing I. ricinus was significantly higher in the urban/suburban habitat (7.2%; 95% CI: 6.1-8.3%) compared to the natural habitat (3.1%; 95% CI: 2.5-3.9%) (χ(2) = 37.451; P < 0.001). Significant local differences in prevalence of infected questing ticks were found among transects within each habitat as well as among years and between seasons. The trapped rodents belonged to six species. Apodemus flavicollis and Myodes glareolus prevailed in both habitats, Microtus arvalis was present only in the natural habitat. I. ricinus comprised 96.3% of the rodent-attached ticks, the rest were Haemaphysalis concinna, Ixodes trianguliceps and Dermacentor reticulatus. Only 0.5% of rodent skin and 0.6% of rodent-attached ticks (only I. ricinus) were infected with A. phagocytophilum. Prevalence of A. phagocytophilum in questing I. ricinus did not correlate significantly with relative abundance of ticks or with abundance of wildlife in the area. CONCLUSION: The study confirms that urban I. ricinus populations are infected with A. phagocytophilum at a higher rate than in a natural habitat of south-western Slovakia and suggests that rodents are not the main reservoirs of the bacterium in the investigated area.

Detection of Murine Herpesvirus 68 (MHV-68) in Dermacentor reticulatus Ticks.

Murid herpesvirus 4 (MuHV 4) strain 68 (MHV-68) is a natural pathogen of murid rodents, which serves as hosts to Dermacentor reticulatus ticks. These ticks are known to transmit multiple pathogens, which can cause diseases in humans and animals. Recently, the detection of MHV-68 antibodies in the blood of animals living in the same biotope as virus-infected mice has suggested the role of ticks in pathogen circulation in nature. Herein, to identify MHV-68 in D. reticulatus ticks, DNA samples from 432 adults were collected at two sites in southwestern Slovakia from 2011 to 2014. Samples were examined by polymerase chain reaction (PCR), targeting ORF50 of MHV-68. Ignoring season and locality, we have found 25.9 % of the male and 44.9 % of the female ticks to be positive. Within ticks collected in Vojka, 40 % (125/312) became positive, at a rate of approximately 6.8 times higher in spring than in autumn (66 vs 9.7 %). In addition, in the spring, 1.4 times more females were positive than males. Within ticks collected in Gabcíkovo, 23.3 % (28/120) became positive, with positive females being twice as frequent. The infecting virus was identified by analyzing amplified products via sequencing and restriction fragment length polymorphism (RFLP) analyses. Using an explantation/co-cultivation procedure, we examined the salivary glands, intestines, and ovaries of five females for live MHV-68. In all organs of two ticks, we identified a virus capable of replication in mammalian cells. This is the first report of MHV-68 detection in D. reticulatus ticks and of a live virus in their organs. Findings encourage further study to determine whether this potential arbovirus, found in salivary glands, is transmissible. It further supports the hypothesis regarding the mediating role of ticks in MHV-68 circulation in nature.

Sexual differences in the sialomes of the zebra tick, Rhipicephalus pulchellus.

Ticks rely exclusively on vertebrate blood for their survival. During feeding ticks inject into their hosts a sophisticated salivary potion that overcomes host hemostasis and adverse inflammatory responses. These mediators may also enhance pathogen transmission. Knowledge of the tick salivary protein repertoire may lead to vaccine targets to disrupt feeding and/or parasite transmission as well as to the discovery of novel pharmacological agents. Male saliva may also assist reproduction because males use their mouthparts to lubricate and introduce their spermatophores into the females' genital pore. The analyses of the sialomes of male and female ticks independently allow us to understand the strategy used by each gender to feed successfully. We sequenced cDNA libraries from pools of salivary glands from adult male and female Rhipicephalus pulchellus feeding at different time points, using the Illumina HiSeq protocol. De novo assembly of a total of 241,229,128 paired-end reads lead to extraction of 50,460 coding sequences (CDS), 11,277 of which had more than 75% coverage to known transcripts, or represented novel sequences, and were submitted to GenBank. Additionally, we generated the proteome, from the salivary gland extracts of male and female R. pulchellus, yielding a total of 454 and 2063 proteins respectively which were identified by one or more peptides with at least 95% confidence. The data set is presented as an annotated hyperlinked Excel spreadsheet, describing 121 putative secreted protein families. Female and male specific transcripts were identified. BIOLOGICAL SIGNIFICANCE: This annotated R. pulchellus database represents a mining field for future experiments involving the resolution of time-dependent transcript expression in this tick species, as well as to define novel vaccine targets and discover novel pharmaceuticals. Gender specific proteins may represent different repertoires of pharmacological reagents to assist feeding by each sex, and in males may represent proteins that assist reproduction similarly to seminal proteins in other animals.

Survival dynamics of tick-borne encephalitis virus in Ixodes ricinus ticks.

Biotic factors contributing to the survival of tick-borne viruses in nature are poorly understood. Using tick-borne encephalitis virus (TBEV) and its principal European vector, Ixodes ricinus, we examined the relative roles of salivary gland infection, co-feeding transmission, and moulting in virus survival. Virus titres in the salivary glands increased after blood-feeding in a time- and dose-dependent manner. This was observed in ticks infected by inoculation but not in ticks infected by the natural route of co-feeding. Amplification of infection prevalence occurred via co-feeding. However, when larvae or nymphs subsequently moulted, the infection prevalence dramatically declined although this was not observed when ticks were infected by inoculation. Trans-stadial survival is a hitherto overlooked parameter that may contribute to the low incidence of TBEV infection in field-collected I. ricinus ticks.

Immunization with recombinant subolesin does not reduce tick infection with tick-borne encephalitis virus nor protect mice against disease.

Tick-borne encephalitis (TBE) is a growing zoonotic disease caused by tick-borne encephalitis virus (TBEV) infection. Although effective vaccines for TBEV are available, on-going vaccination efforts are insufficient to prevent increase in TBE cases annually. Vaccination with arthropod vector antigens to reduce vector infestations and vector capacity allows control of several vector-borne diseases by targeting their common vector. Subolesin (SUB) is a tick protective antigen that has a role in tick innate immunity and other molecular pathways and has been shown to protect against tick infestations and infection by vector-borne pathogens. However, SUB expression and the effect of SUB immunization have not been evaluated for tick-borne viruses. Herein, we showed that SUB expression is downregulated during Ixodes ricinus tick feeding but induced in ticks infected with TBEV, thus supporting a role for this molecule in tick innate immune response to virus infection. Immunization with recombinant SUB reduced SUB mRNA levels in nymphs co-feeding with infected females and suggested and effect on tick infestations in mice. However, SUB immunization did not reduce tick infection with TBEV nor protect mice against TBE. These results suggested that SUB is not a good candidate antigen for vaccination against TBEV and support the characterization of tick-pathogen interactions to identify mechanisms that could be targeted to reduce TBEV infection and transmission by ticks.

Evaluation of chimeric yellow fever 17D/dengue viral replication in ticks.

Chimeric yellow fever 17D/DENV-1-4 viruses (CYD-1-4) have been developed as a tetravalent dengue vaccine candidate which is currently being evaluated in efficacy trials in Asia and America. While YF 17D and DENV are mosquito-borne flaviviruses, it has been shown that CYD-1-4 do not replicate after oral infection in mosquitoes and are not transmitted to new hosts. To further document the risk of environmental dissemination of these viruses, we evaluated the replication of CYD-1-4 in ticks, the vector of tick-borne encephalitis virus (TBEV), another member of the flavivirus family. Females of two hard tick species, Ixodes ricinus and Rhipicephalus appendiculatus, were inoculated intracoelomically with CYD-1-4 viruses and parent viruses (DENV-1-4 and YF 17D). Virus persistence and replication was assessed 2, 16, and 44 days post-inoculation by plaque titration and qRT-PCR. CYD-1-4 viruses were detected in I. ricinus ticks at early time points post-inoculation, but with infectious titers at least 100-fold lower than those observed in TBEV-infected ticks. Unlike TBEV, complete viral clearance occurred by day 44 in most ticks except for CYD-2, which had a tendency to decline. In addition, while about 70% of TBEV-infected I. ricinus nymphs acquired infection by co-feeding with infected tick females on non-viremic hosts, no co-feeding transmission of CYD-2 virus was detected. Based on these results, we conclude that the risk of dissemination of the candidate vaccine viruses by tick bite is highly unlikely.