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Ticks are the main vectors for the transmission of bacterial, protist and viral pathogens in Europe affecting wildlife and domestic animals. However, some of them are zoonotic and can cause serious, sometimes fatal, problems in human health. A systematic review in PubMed/MEDLINE database was conducted to determine the spatial distribution and host and tick species ranges of a selection of tick-borne bacteria (Anaplasma spp., Borrelia spp., Coxiella spp., and Rickettsia spp.), protists (Babesia spp. and Theileria spp.), and viruses (Orthonairovirus, and flaviviruses tick-borne encephalitis virus and louping ill virus) on the European continent in a five-year period (November 2017 - November 2022). Only studies using PCR methods were selected, retrieving a total of 429 articles. Overall, up to 85 species of the selected tick-borne pathogens were reported from 36 European countries, and Anaplasma spp. was described in 37% (159/429) of the articles, followed by Babesia spp. (34%, 148/429), Borrelia spp. (34%, 147/429), Rickettsia spp. (33%, 142/429), Theileria spp. (11%, 47/429), tick-borne flaviviruses (9%, 37/429), Orthonairovirus (7%, 28/429) and Coxiella spp. (5%, 20/429). Host and tick ranges included 97 and 50 species, respectively. The highest tick-borne pathogen diversity was detected in domestic animals, and 12 species were shared between humans, wildlife, and domestic hosts, highlighting the following zoonotic species: Anaplasma phagocytophilum, Babesia divergens, Babesia microti, Borrelia afzelii, Borrelia burgdorferi s.s., Borrelia garinii, Borrelia miyamotoi, Crimean-Congo hemorrhagic fever virus, Coxiella burnetii, Rickettsia monacensis and tick-borne encephalitis virus. These results contribute to the implementation of effective interventions for the surveillance and control of tick-borne diseases., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Paratransgenesis through genetic manipulation of symbiotic or commensal microorganisms has been proposed as an effective and environmentally sound approach for the control of vector-borne diseases, including tick bite-related pathologies, and reducing pathogen transmission. Here, we present a protocol for Sphingomonas transformation with Anaplasma phagocytophilum major surface protein 4 and heat shock protein 70. We describe a step-by-step protocol for in vitro study of interactions between transformed Franken Sphingomonas and Ixodes scapularis ISE6 tick cells during A. phagocytophilum infection. For complete details on the use and execution of this protocol, please refer to Mazuecos et al. (2023). 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Lyme borreliosis (LB) caused by Borrelia burgdorferi sensu lato complex spirochetes is one of the tick-borne diseases with high prevalence and social/economic burden in the United States, Spain, and other European countries. The objective is to address limited information available about the incidence, prevalence, and symptoms of LB, current prevention, and treatment interventions that are not adequately focused and thus not very effective against this disease. Methods: To address these limitations, in this study, we used a citizen science approach to evaluate the LB-associated risks and implementation of control interventions in Spain. A total of 405 participants in the survey were included in the analysis. Responses to the questionnaire were received during January-July 2022. The questionnaire contained qualitative and quantitative questions. Homogeneity among binary variables was analyzed using a Fisher's exact test. Results: Despite limitations of the study associated with response to the questionnaire and information on tick species, the results evidenced the effect of factors such as age, gender, tick bites, disease clinical signs, comorbidities such as alpha-gal syndrome, health care services, and treatment effectiveness affecting LB. Conclusions: The main conclusions of the study highlight the need for better surveillance of tick infestations, pathogen infection, and diagnosis of LB and related comorbidities. To advance in disease prevention, diagnosis, and treatment, new interventions need to be developed and implemented in both public and private health care services. Providing access to these results to the society, health care system, and scientists is important to further advance in disease surveillance, diagnosis, control, and prevention.

Background: Alpha-Gal syndrome (AGS) is a tick-borne food allergy caused by IgE antibodies against the glycan galactose-alpha-1,3-galactose (α-Gal) present in glycoproteins and glycolipids from mammalian meat. To advance in the diagnosis and treatment of AGS, further research is needed to unravel the molecular and immune mechanisms underlying this syndrome. The objective of this study is the characterization of tick salivary components and proteins with and without α-Gal modifications involved in modulating human immune response against this carbohydrate., Methods: Protein and α-Gal content were determined in tick saliva components, and proteins were identified by proteomics analysis of tick saliva fractions. Pathophysiological changes were recorded in the zebrafish (Danio rerio) model after exposure to distinct Ixodes ricinus tick salivary components. Serum samples were collected from zebrafish at day 8 of exposure to determine anti-α-Gal, anti-glycan, and anti-tick saliva protein IgM antibody titers by enzyme-linked immunosorbent assay (ELISA)., Results: Zebrafish treated with tick saliva and saliva protein fractions combined with non-protein fractions demonstrated significantly higher incidence of hemorrhagic type allergic reactions, abnormal behavioral patterns, or mortality when compared to the phosphate-buffered saline (PBS)-treated control group. The main tick salivary proteins identified in these fractions with possible functional implication in AGS were the secreted protein B7P208-salivary antigen p23 and metalloproteases. Anti-α-Gal and anti-tick salivary gland IgM antibody titers were significantly higher in distinct saliva protein fractions and deglycosylated saliva group when compared with PBS-treated controls. Anti-glycan antibodies showed group-related profiles., Conclusions: Results support the hypothesis that tick salivary biomolecules with and without α-Gal modifications are involved in modulating immune response against this carbohydrate., (© 2023. The Author(s).)

Ticks attaching to ear canals of humans and animals are the cause of otoacariasis, common in rural areas of Nepal. The plant Clerodendrum viscosum is used in multiple indigenous systems of medicine by ethnic communities in the Indo-Nepali-Malaysian region. Visiting the Chitwan National Park, we learned that in indigenous medicine, flower extract of C. viscosum is utilized to treat digestive disorders and extracts from leaves as tick repellent to prevent ticks from invading or to remove them from the ear canal. The objective of our study was to provide support to indigenous medicine by characterizing the in vivo effect of leave extracts on ticks under laboratory conditions and its phytochemical composition. We collected plant parts of C. viscosum (leaves and flowers) and mango (Mangifera indica) leaves at the Chitwan National Park, previously associated with repellent activity to characterize their effect on Ixodes ricinus ticks by in vivo bioassays. A Q-ToF high-resolution analysis (HPLC-ESI-QToF) was conducted to elucidate phenolic compounds with potential repellent activity. Clerodendrum viscosum and M. indica leaf extracts had the highest tick repellent efficacy (%E = 80-100%) with significant differences when compared to C. viscosum flowers extracts (%E = 20-60%) and phosphate-buffered saline. Phytochemicals with tick repellent function as caffeic acid, fumaric acid and p-coumaric acid glucoside were identified in C. viscosum leaf extracts by HPLC-ESI-QToF, but not in non-repellent flower extracts. These results support the Nepali indigenous medicine application of C. viscosum leaf extracts to repel ticks. Additional research is needed for the development of natural and green repellent formulations to reduce the risks associated with ticks resistant to acaricides., (© 2023. The Author(s).)

Ticks are arthropod vectors that cause disease by pathogen transmission and whose bites could be related to allergic reactions impacting human health worldwide. In some individuals, high levels of immunoglobulin E antibodies against the glycan Galα1-3Galβ1-(3)4GlcNAc-R (α-Gal) have been induced by tick bites. Anaphylactic reactions mediated by glycoproteins and glycolipids containing the glycan α-Gal, present in tick saliva, are related to alpha-Gal syndrome (AGS) or mammalian meat allergy. Zebrafish (Danio rerio) has become a widely used vertebrate model for the study of different pathologies. In this study, zebrafish was used as a model for the study of allergic reactions in response to α-Gal and mammalian meat consumption because, like humans, they do not synthesize this glycan. For this purpose, behavioral patterns and hemorrhagic anaphylactic-type allergic reactions in response to Ixodes ricinus tick saliva and mammalian meat consumption was evaluated. This experimental approach allows the obtention of valid data that support the zebrafish animal model for the study of tick-borne allergies including AGS.

The lack of tools for the precise manipulation of the tick microbiome is currently a major limitation to achieve mechanistic insights into the tick microbiome. Anti-tick microbiota vaccines targeting keystone bacteria of the tick microbiota alter tick feeding, but their impact on the taxonomic and functional profiles of the tick microbiome has not been tested. In this study, we immunized a vertebrate host model ( Mus musculus ) with live bacteria vaccines targeting keystone (i.e., Escherichia-Shigella ) or non-keystone (i.e., Leuconostoc ) taxa of tick microbiota and tested the impact of bacterial-specific antibodies (Abs) on the structure and function of tick microbiota. We also investigated the effect of these anti-microbiota vaccines on mice gut microbiota composition. Our results showed that the tick microbiota of ticks fed on Escherichia coli -immunized mice had reduced Escherichia-Shigella abundance and lower species diversity compared to ticks fed on control mice immunized with a mock vaccine. Immunization against keystone bacteria restructured the hierarchy of nodes in co-occurrence networks and reduced the resistance of the bacterial network to taxa removal. High levels of E. coli -specific IgM and IgG were negatively correlated with the abundance of Escherichia-Shigella in tick microbiota. These effects were not observed when Leuconostoc was targeted with vaccination against Leuconostoc mesenteroides . Prediction of functional pathways in the tick microbiome using PICRUSt2 revealed that E. coli vaccination reduced the abundance of lysine degradation pathway in tick microbiome, a result validated by qPCR. In contrast, the gut microbiome of immunized mice showed no significant alterations in the diversity, composition and abundance of bacterial taxa. Our results demonstrated that anti-tick microbiota vaccines are a safe, specific and an easy-to-use tool for manipulation of vector microbiome. These results guide interventions for the control of tick infestations and pathogen infection/transmission., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mateos-Hernández, Obregón, Wu-Chuang, Maye, Bornères, Versillé, de la Fuente, Díaz-Sánchez, Bermúdez-Humarán, Torres-Maravilla, Estrada-Peña, Hodžić, Šimo and Cabezas-Cruz.)

Ticks (Acari: Ixodidae) are considered to be the most important vectors of disease-causing pathogens in domestic and wild animals, and emerging and re-emerging tick-borne diseases (TBD) exert an enormous impact on them. Wild ungulates are hosts for a wide variety of tick species and tick-borne pathogens that affect human and animal health. Consequently, the control of tick infestations and tick-borne pathogen prevalence is essential in some regions. Acaricides and animal management or culling have been used for the control of tick infestations and TBD, but tick vaccines constitute the best alternative to reduce the impact of acaricides on tick resistance and the environment. Previous results of controlled vaccination trials have shown that the Q38 Subolesin/Akirin chimera containing conserved protective epitopes could be a candidate universal antigen to control multiple tick species infestations. Thus, vaccination trials are necessary to validate these results under field conditions. In this study, we characterized the effect of Q38 vaccine on a wild population of European roe deer (Capreolus capreolus) in the Andalusian roe deer Reference Station (Junta de Andalucía, Cádiz, Spain). In this location, roe deer suffer especially severe parasitic conditions in some periods and commercial pesticides and ixodicides that are authorized to control ticks without specificity are frequently applied in the field, posing a threat to the environment. Animals vaccinated over a three-year period showed an antibody response to the vaccine antigen and a reduction in tick infestations by multiple species including Hyalomma marginatum, H. lusitanicum, Rhipicephalus bursa and Ixodes ricinus previously identified in roe deer, when compared to untreated controls. These results suggest the efficacy of Q38 for the control of tick infestations in wildlife., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Aerobic organisms evolved conserved mechanisms controlling the generation of reactive oxygen species (ROS) to maintain redox homeostasis signaling and modulate signal transduction, gene expression and cellular functional responses under physiological conditions. The production of ROS by mitochondria is essential in the oxidative stress associated with different pathologies and in response to pathogen infection. Anaplasma phagocytophilum is an intracellular pathogen transmitted by Ixodes scapularis ticks and causing human granulocytic anaplasmosis. Bacteria multiply in vertebrate neutrophils and infect first tick midgut cells and subsequently hemocytes and salivary glands from where transmission occurs. Previous results demonstrated that A. phagocytophilum does not induce the production of ROS as part of its survival strategy in human neutrophils. However, little is known about the role of ROS during pathogen infection in ticks. In this study, the role of tick oxidative stress during A. phagocytophilum infection was characterized through the function of different pathways involved in ROS production. The results showed that tick cells increase mitochondrial ROS production to limit A. phagocytophilum infection, while pathogen inhibits alternative ROS production pathways and apoptosis to preserve cell fitness and facilitate infection. The inhibition of NADPH oxidase-mediated ROS production by pathogen infection appears to occur in both neutrophils and tick cells, thus supporting that A. phagocytophilum uses common mechanisms for infection of ticks and vertebrate hosts. However, differences in ROS response to A. phagocytophilum infection between human and tick cells may reflect host-specific cell tropism that evolved during pathogen life cycle.

The microRNAs (miRNAs) are a class of small noncoding RNAs that have important regulatory roles in multicellular organisms including innate and adaptive immune pathways to control bacterial, parasite and viral infections, and pathogens could modify host miRNA profile to facilitate infection and multiplication. Therefore, understanding the function of host miRNAs in response to pathogen infection is relevant to characterize host-pathogen molecular interactions and to provide new targets for effective new interventions for the control infectious diseases. The objective of this study was to characterize the dynamics and functional significance of the miRNA response of the tick vector Ixodes scapularis in response to Anaplasma phagocytophilum infection, the causative agent of human and animal granulocytic anaplasmosis. To address this objective, the composition of tick miRNAs, functional annotation, and expression profiling was characterized using high throughout RNA sequencing in uninfected and A. phagocytophilum-infected I. scapularis ISE6 tick cells, a model for tick hemocytes involved in pathogen infection. The results provided new evidences on the role of tick miRNA during pathogen infection, and showed that A. phagocytophilum modifies I. scapularis tick cell miRNA profile and upregulates isc-mir-79 to facilitate infection by targeting the Roundabout protein 2 (Robo2) pathway. Furthermore, these results suggested new targets for interventions to control pathogen infection in ticks.

Exploring the microbial diversity of ticks is crucial to understand geographical dispersion and pathogen transmission. Tick microbes participate in many biological processes implicated in the acquisition, maintenance, and transmission of pathogens, and actively promote host phenotypic changes, and adaptation to new environments. The microbial community of Ixodes ventalloi still remains unexplored. In this study, the bacterial microbiota of wild-caught I. ventalloi was characterized using shotgun-metagenomic sequencing in samples from unfed adults collected during December 2013-January 2014 in two locations from Sicily, Italy. The microbiota identified in I. ventalloi was mainly composed of symbiotic, commensal, and environmental bacteria. Interestingly, we identified the genera Anaplasma and Borrelia as members of the microbiota of I. ventalloi. These results advance our information on I. ventalloi microbiota composition, with potential implications in tick-host adaptation, geographic expansion, and vector competence., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

The carbohydrate Galα1-3Galβ1-(3)4GlcNAc-R (α-Gal) is produced in all mammals except for humans, apes and old world monkeys that lost the ability to synthetize this carbohydrate. Therefore, humans can produce high antibody titers against α-Gal. Anti-α-Gal IgE antibodies have been associated with tick-induced allergy (i.e. α-Gal syndrome) and anti-α-Gal IgG/IgM antibodies may be involved in protection against malaria, leishmaniasis and Chagas disease. The α-Gal on tick salivary proteins plays an important role in the etiology of the α-Gal syndrome. However, whether ticks are able to produce endogenous α-Gal remains currently unknown. In this study, the Ixodes scapularis genome was searched for galactosyltransferases and three genes were identified as potentially involved in the synthesis of α-Gal. Heterologous gene expression in α-Gal-negative cells and gene knockdown in ticks confirmed that these genes were involved in α-Gal synthesis and are essential for tick feeding. Furthermore, these genes were shown to play an important role in tick-pathogen interactions. Results suggested that tick cells increased α-Gal levels in response to Anaplasma phagocytophilum infection to control bacterial infection. These results provided the molecular basis of endogenous α-Gal production in ticks and suggested that tick galactosyltransferases are involved in vector development, tick-pathogen interactions and possibly the etiology of α-Gal syndrome in humans.

An innovative metaomics approach integrating metatranscriptomics and metaproteomics was used to characterize bacterial communities in the microbiota of the Lyme borreliosis spirochete vector, Ixodes ricinus (Acari: Ixodidae). Whole internal tissues and salivary glands from unfed larvae and female ticks, respectively were used. Reused I. ricinus RNA-sequencing data for metranscriptomics analysis together with metaproteomics provided a better characterization of tick bacterial microbiota by increasing bacteria identification and support for identified bacteria with putative functional implications. The results showed the presence of symbiotic, commensal, soil, environmental, and pathogenic bacteria in the I. ricinus microbiota, including previously unrecognized commensal and soil microorganisms. The results of the metaomics approach may have implications in the characterization of putative mechanisms by which pathogen infection manipulates tick microbiota to facilitate infection. Metaomics approaches integrating different omics datasets would provide a better description of tick microbiota compositions, and insights into tick interactions with microbiota, pathogens and hosts., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Malaria is a mosquito-borne disease affecting millions of people mainly in Sub-Saharan Africa, Asia and some South American countries. Drug resistance to first-line antimalarial drugs (e.g. chloroquine, sulfadoxine-pyrimethamine and artemisinin) is a major constrain in malaria control. Antimicrobial peptides (AMPs) have shown promising results in controlling Plasmodium spp. parasitemia in in vitro and in vivo models of infection. Defensins are AMPs that act primarily by disrupting the integrity of cell membranes of invasive microbes. We previously showed that defensins from the tick Ixodes ricinus inhibited significantly the growth of P. falciparum in vitro, a property that was conserved during evolution. Here, we tested the activity of three I. ricinus defensins against P. chabaudi in mice. A single dose of defensin (120 μl of 1 mg/ml solution) was administered intravenously to P. chabaudi-infected mice, and the parasitemia was followed for 24 h post-treatment. Defensin treatment inhibited significantly the replication (measured as increases in parasitemia) of P. chabaudi after 1 h and 12 h of treatment. Furthermore, defensin injection was not associated with toxicity. These results agreed with the previous report of antiplasmodial activity of tick defensins against P. falciparum in vitro and justify further studies for the use of tick defensins to control malaria., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Ticks, pathogens, and vertebrates interact in a background of environmental features that regulate the densities of ticks and vertebrates, affecting their contact rates and thence the circulation of the pathogens. Regional scale studies are invaluable sources of information about the regulation of these interactions, but a large-scale analysis of the interaction of communities of ticks, hosts, and the environment has been never modeled. This study builds on network analysis, satellite-derived climate and vegetation, and environmental modeling, quantifying the interactions between the tick Ixodes ricinus and the transmitted bacteria of the complex Borrelia burgdorferi s.l. in the Western Palaearctic. We derived the rates of contact of the tick with 162 species of vertebrates recorded as hosts, and the relative importance of each vertebrate in the circulation of the pathogen. We compiled more than 11 millions of pairs of coordinates of the vertebrates, deriving distribution models of each species and the relative faunal composition in the target territory. The results of the modeling of the distribution of the tick and its hosts, weighted by their importance in the circulation of Borrelia captured the spatial patterns of interactions that allow the circulation of the pathogen. Results indicate that both I. ricinus and B. burgdorferi s.l. are supported in the Western Palaearctic by complex communities of vertebrates, which have large distribution ranges. This high functional redundancy results in the pervasiveness of B. burgdorferi s.l., which depends on the gradient of contributions of the large community of vertebrates, instead of relying on a few dominant vertebrates, which was the prevailing paradigm. Most prominent reservoirs of the pathogen are distributed in specific regions of the environmental niche. However, literally dozens of potential reservoirs can colonize many other environmental regions, marginally but efficiently contributing to the circulation of the pathogen. These results consistently point to the need of evaluating the beta-diversity of the community of vertebrates acting as reservoirs of the pathogen to better know the interactions with the vector. They also demonstrate why the pathogen is so resilient to perturbations in the composition of the reservoirs.

The obligate intracellular pathogen, Anaplasma phagocytophilum , is the causative agent of life-threatening diseases in humans and animals. A. phagocytophilum is an emerging tick-borne pathogen in the United States, Europe, Africa and Asia, with increasing numbers of infected people and animals every year. It is increasingly recognized that intracellular pathogens modify host cell metabolic pathways to increase infection and transmission in both vertebrate and invertebrate hosts. Recent reports have shown that amino acids are central to the host-pathogen metabolic interaction. In this study, a genome-wide search for components of amino acid metabolic pathways was performed in Ixodes scapularis , the main tick vector of A. phagocytophilum in the United States, for which the genome was recently published. The enzymes involved in the synthesis and degradation pathways of the twenty amino acids were identified. Then, the available transcriptomics and proteomics data was used to characterize the mRNA and protein levels of I. scapularis amino acid metabolic pathway components in response to A. phagocytophilum infection of tick tissues and ISE6 tick cells. Our analysis was focused on the interplay between carbohydrate and amino acid metabolism during A. phagocytophilum infection in ISE6 cells. The results showed that tick cells increase the synthesis of phosphoenolpyruvate (PEP) from tyrosine to control A. phagocytophilum infection. Metabolic pathway analysis suggested that this is achieved by (i) increasing the transcript and protein levels of mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M), (ii) shunting tyrosine into the tricarboxylic acid (TCA) cycle to increase fumarate and oxaloacetate which will be converted into PEP by PEPCK-M, and (iii) blocking all the pathways that use PEP downstream gluconeogenesis (i.e., de novo serine synthesis pathway (SSP), glyceroneogenesis and gluconeogenesis). While sequestering host PEP may be critical for this bacterium because it cannot actively carry out glycolysis to produce PEP, excess of this metabolite may be toxic for A. phagocytophilum . The present work provides a more comprehensive view of the major amino acid metabolic pathways involved in the response to pathogen infection in ticks, and provides the basis for further studies to develop novel strategies for the control of granulocytic anaplasmosis.

Anaplasma phagocytophilum is an emerging tick-borne pathogen causing human granulocytic anaplasmosis (HGA), tick-borne fever (TBF) in small ruminants, and other forms of anaplasmosis in different domestic and wild animals. The main vectors of this pathogen are Ixodes tick species, particularly I. scapularis in the United States and I. ricinus in Europe. One of the main limitations for the development of effective vaccines for the prevention and control of A. phagocytophilum infection and transmission is the identification of effective tick protective antigens. The objective of this study was to apply a vaccinomics approach to I. scapularis - A. phagocytophilum interactions for the identification and characterization of candidate tick protective antigens for the control of vector infestations and A. phagocytophilum infection. The vaccinomics pipeline included the use of quantitative transcriptomics and proteomics data from uninfected and A. phagocytophilum -infected I. scapularis ticks for the selection of candidate protective antigens based on the variation in tick mRNA and protein levels in response to infection, their putative biological function, and the effect of antibodies against these proteins on tick cell apoptosis and pathogen infection. The characterization of selected candidate tick protective antigens included the identification and characterization of I. ricinus homologs, functional characterization by different methodologies including RNA interference, immunofluorescence, gene expression profiling, and artificial tick feeding on rabbit antibodies against the recombinant antigens to select the candidates for vaccination trials. The vaccinomics pipeline developed in this study resulted in the identification of two candidate tick protective antigens that could be selected for future vaccination trials. The results showed that I. scapularis lipocalin (ISCW005600) and lectin pathway inhibitor (AAY66632) and I. ricinus homologs constitute candidate protective antigens for the control of vector infestations and A. phagocytophilum infection. Both antigens are involved in the tick evasion of host defense response and pathogen infection and transmission, but targeting different immune response pathways. The vaccinomics pipeline proposed here could be used to continue the identification and characterization of candidate tick protective antigens for the development of effective vaccines for the prevention and control of HGA, TBF, and other forms of anaplasmosis caused by A. phagocytophilum .

The obligate intracellular pathogen Anaplasma phagocytophilum infects vertebrate and tick hosts. In this study, a genome-wide search for cytoskeleton components was performed in the tick vector, Ixodes scapularis . The available transcriptomics and proteomics data was then used to characterize the mRNA and protein levels of I. scapularis cytoskeleton components in response to A. phagocytophilum infection. The results showed that cytoskeleton components described in other model organisms were present in the I. scapularis genome. One type of intermediate filaments (lamin), a family of septins that was recently implicated in the cellular response to intracellular pathogens, and several members of motor proteins (kinesins and dyneins) that could be implicated in the cytoplasmic movements of A. phagocytophilum were found. The results showed that levels of tubulin, actin, septin, actin-related proteins and motor proteins were affected by A. phagocytophilum , probably to facilitate infection in I. scapularis . Functional studies demonstrated a role for selected cytoskeleton components in pathogen infection. These results provided a more comprehensive view of the cytoskeletal components involved in the response to A. phagocytophilum infection in ticks.

Background: Tick-borne diseases greatly impact human and animal health worldwide, and vaccines are an environmentally friendly alternative to acaricides for their control. Recent results have suggested that aquaporin (AQP) water channels have a key function during tick feeding and development, and constitute good candidate antigens for the control of tick infestations., Methods: Here we describe the effect of vaccination with the Ixodes ricinus AQP1 (IrAQP) and a tick AQP conserved region (CoAQP) on I. ricinus tick larval mortality, feeding and molting., Results: We demonstrated that vaccination with IrAQP and CoAQP had an efficacy of 32% and 80%, respectively on the control of I. ricinus larvae by considering the cumulative effect on reducing tick survival and molting., Conclusions: The effect of the AQP vaccines on larval survival and molting is essential to reduce tick infestations, and extended previous results on the effect of R. microplus AQP1 on the control of cattle tick infestations. These results supports that AQP, and particularly CoAQP, might be a candidate protective antigen for the control of different tick species., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: There has been an emergence and expansion of tick-borne diseases in Europe, Asia and North America in recent years, including Lyme disease, tick-borne encephalitis and human anaplasmosis. The primary vectors implicated are hard ticks of the genus Ixodes. Although much is known about the host response to these bacterial and viral pathogens, there is limited knowledge of the cellular responses to infection within the tick vector. The bacterium Anaplasma phagocytophilum is able to bypass apoptotic processes in ticks, enabling infection to proceed. However, the tick cellular responses to infection with the flaviviruses tick-borne encephalitis virus (TBEV) and louping ill virus (LIV), which cause tick-borne encephalitis and louping ill respectively, are less clear., Results: Infection and transcriptional analysis of the Ixodes ricinus tick cell line IRE/CTVM20 with the viruses LIV and TBEV, and the bacterium A. phagocytophilum, identified activation of common and distinct cellular pathways. In particular, commonly-upregulated genes included those that modulate apoptotic pathways, putative anti-pathogen genes, and genes that influence the tick innate immune response, including selective activation of toll genes., Conclusion: These data provide an insight into potential key genes involved in the tick cellular response to viral or bacterial infection, which may promote cell survival and host resistance.

The insect immune deficiency (IMD) pathway resembles the tumour necrosis factor receptor network in mammals and senses diaminopimelic-type peptidoglycans present in Gram-negative bacteria. Whether unidentified chemical moieties activate the IMD signalling cascade remains unknown. Here, we show that infection-derived lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) and 1-palmitoyl-2-oleoyl diacylglycerol (PODAG) stimulate the IMD pathway of ticks. The tick IMD network protects against colonization by three distinct bacteria, that is the Lyme disease spirochete Borrelia burgdorferi and the rickettsial agents Anaplasma phagocytophilum and A. marginale. Cell signalling ensues in the absence of transmembrane peptidoglycan recognition proteins and the adaptor molecules Fas-associated protein with a death domain (FADD) and IMD. Conversely, biochemical interactions occur between x-linked inhibitor of apoptosis protein (XIAP), an E3 ubiquitin ligase, and the E2 conjugating enzyme Bendless. We propose the existence of two functionally distinct IMD networks, one in insects and another in ticks.

The obligate intracellular pathogen, Anaplasma phagocytophilum , is the causative agent of human, equine, and canine granulocytic anaplasmosis and tick-borne fever (TBF) in ruminants. A. phagocytophilum has become an emerging tick-borne pathogen in the United States, Europe, Africa, and Asia, with increasing numbers of infected people and animals every year. It has been recognized that intracellular pathogens manipulate host cell metabolic pathways to increase infection and transmission in both vertebrate and invertebrate hosts. However, our current knowledge on how A. phagocytophilum affect these processes in the tick vector, Ixodes scapularis is limited. In this study, a genome-wide search for components of major carbohydrate metabolic pathways was performed in I. scapularis ticks for which the genome was recently published. The enzymes involved in the seven major carbohydrate metabolic pathways glycolysis, gluconeogenesis, pentose phosphate, tricarboxylic acid cycle (TCA), glyceroneogenesis, and mitochondrial oxidative phosphorylation and β-oxidation were identified. Then, the available transcriptomics and proteomics data was used to characterize the mRNA and protein levels of I. scapularis major carbohydrate metabolic pathway components in response to A. phagocytophilum infection of tick tissues and cultured cells. The results showed that major carbohydrate metabolic pathways are conserved in ticks. A. phagocytophilum infection inhibits gluconeogenesis and mitochondrial metabolism, but increases the expression of glycolytic genes. A model was proposed to explain how A. phagocytophilum could simultaneously control tick cell glucose metabolism and cytoskeleton organization, which may be achieved in part by up-regulating and stabilizing hypoxia inducible factor 1 alpha in a hypoxia-independent manner. The present work provides a more comprehensive view of the major carbohydrate metabolic pathways involved in the response to A. phagocytophilum infection in ticks, and provides the basis for further studies to develop novel strategies for the control of granulocytic anaplasmosis.

As tick-borne diseases are on the rise, an international effort resulted in the sequence and assembly of the first genome of a tick vector. This result promotes research on comparative, functional and evolutionary genomics and the study of tick-host-pathogen interactions to improve human, animal and ecosystem health on a global scale.

Background: The intracellular bacteria Anaplasma phagocytophilum are emerging zoonotic pathogens affecting human and animal health, and a good model for the study of tick-host-pathogen interactions. This tick-borne pathogen is transmitted by Ixodes scapularis in the United States where it causes human granulocytic anaplasmosis. Tick midguts and salivary glands play a major role during tick feeding and development, and in pathogen acquisition, multiplication and transmission. Vertebrate host proteins are found in tick midguts after feeding and have been described in the salivary glands of fed and unfed ticks, suggesting a role for these proteins during tick feeding and development. Furthermore, recent results suggested the hypothesis that pathogen infection affects tick metabolic processes to modify host protein digestion and persistence in the tick with possible implications for tick physiology and pathogen life-cycle., Methods: To address this hypothesis, herein we used I. scapularis female ticks fed on uninfected and A. phagocytophilum-infected sheep to characterize host protein content in midguts and salivary glands by proteomic analysis of tick tissues., Results: The results evidenced a clear difference in the host protein content between tick midguts and salivary glands in response to infection suggesting that A. phagocytophilum selectively manipulates the levels of vertebrate host proteins in ticks in a tissue-specific manner to facilitate pathogen infection, multiplication and transmission while preserving tick feeding and development. The mechanisms by which A. phagocytophilum manipulates the levels of vertebrate host proteins are not known, but the results obtained here suggested that it might include the modification of proteolytic pathways., Conclusions: The results of this study provided evidence to support that A. phagocytophilum affect tick proteolytic pathways to selectively manipulate the levels of vertebrate host proteins in a tissue-specific manner to increase tick vector capacity. Investigating the biological relevance of host proteins in tick biology and pathogen infection and the mechanisms used by A. phagocytophilum to manipulate host protein content is essential to advance our knowledge of tick-host-pathogen molecular interactions. These results have implications for the identification of new targets for the development of vaccines for the control of tick-borne diseases.

Diseases transmitted by ticks greatly impact human and animal health and their control is important for the eradication of tick-borne diseases. Vaccination is an environmentally friendly alternative for tick control. Recent results have suggested that Subolesin/Akirin (SUB/AKR) are good candidate antigens for the control of arthropod vector infestations. Here, we describe the effect of vaccination with the Q38 chimera containing SUB/AKR conserved protective epitopes on Ixodes ricinus and Dermacentor reticulatus tick larval mortality, feeding and molting. We demonstrated that Q38 vaccination had an efficacy of 99.9% and 46.4% on the control of I. ricinus and D. reticulatus larvae by considering the cumulative effect on reducing tick survival and molting. The effect of the Q38 vaccine on larval feeding and molting is essential to reduce tick infestations and supports that Q38 might be a candidate universal antigen for the control of multiple tick species that can infest the same host., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Anaplasma phagocytophilum are transmitted by Ixodes spp. ticks and have become one of the most common and relevant tick-borne pathogens due to their impact on human and animal health. Recent results have increased our understanding of the molecular interactions between Ixodes scapularis and A. phagocytophilum through the demonstration of tissue-specific molecular pathways that ensure pathogen infection, development and transmission by ticks. However, little is known about the Ixodes ricinus genes and proteins involved in the response to A. phagocytophilum infection. The tick species I. scapularis and I. ricinus are evolutionarily closely related and therefore similar responses are expected in A. phagocytophilum-infected cells. However, differences may exist between I. scapularis ISE6 and I. ricinus IRE/CTVM20 tick cells associated with tissue-specific signatures of these cell lines. To address this hypothesis, the transcriptional response to A. phagocytophilum infection was characterized by RNA sequencing and compared between I. scapularis ISE6 and I. ricinus IRE/CTVM20 tick cell lines. The transcriptional response to infection of I. scapularis ISE6 cells resembled that of tick hemocytes while the response in I. ricinus IRE/CTVM20 cells was more closely related to that reported previously in infected tick midguts. The inhibition of cell apoptosis by A. phagocytophilum appears to be a key adaptation mechanism to facilitate infection of both vertebrate and tick cells and was used to investigate further the tissue-specific response of tick cell lines to pathogen infection. The results supported a role for the intrinsic pathway in the inhibition of cell apoptosis by A. phagocytophilum infection of I. scapularis ISE6 cells. In contrast, the results in I. ricinus IRE/CTVM20 cells were similar to those obtained in tick midguts and suggested a role for the JAK/STAT pathway in the inhibition of apoptosis in tick cells infected with A. phagocytophilum. Nevertheless, tick cell lines were derived from embryonated eggs and may contain various cell populations with different morphology and behavior that could affect transcriptional response to infection. These results suggested tissue-specific signatures in I. scapularis ISE6 and I. ricinus IRE/CTVM20 tick cell line response to A. phagocytophilum infection that support their use as models for the study of tick-pathogen interactions.

Ticks transmit more pathogens to humans and animals than any other arthropod. We describe the 2.1 Gbp nuclear genome of the tick, Ixodes scapularis (Say), which vectors pathogens that cause Lyme disease, human granulocytic anaplasmosis, babesiosis and other diseases. The large genome reflects accumulation of repetitive DNA, new lineages of retro-transposons, and gene architecture patterns resembling ancient metazoans rather than pancrustaceans. Annotation of scaffolds representing ∼57% of the genome, reveals 20,486 protein-coding genes and expansions of gene families associated with tick-host interactions. We report insights from genome analyses into parasitic processes unique to ticks, including host 'questing', prolonged feeding, cuticle synthesis, blood meal concentration, novel methods of haemoglobin digestion, haem detoxification, vitellogenesis and prolonged off-host survival. We identify proteins associated with the agent of human granulocytic anaplasmosis, an emerging disease, and the encephalitis-causing Langat virus, and a population structure correlated to life-history traits and transmission of the Lyme disease agent.

Background: Ixodid ticks are important vectors of a wide variety of viral, bacterial and protozoan pathogens of medical and veterinary importance. Although several studies have elucidated tick responses to bacteria, little is known about the tick response to viruses. To gain insight into the response of tick cells to flavivirus infection, the transcriptomes and proteomes of two Ixodes spp cell lines infected with the flavivirus tick-borne encephalitis virus (TBEV) were analysed., Methods: RNA and proteins were isolated from the Ixodes scapularis-derived cell line IDE8 and the Ixodes ricinus-derived cell line IRE/CTVM19, mock-infected or infected with TBEV, on day 2 post-infection (p.i.) when virus production was increasing, and on day 6 p.i. when virus production was decreasing. RNA-Seq and mass spectrometric technologies were used to identify changes in abundance of, respectively, transcripts and proteins. Functional analyses were conducted on selected transcripts using RNA interference (RNAi) for gene knockdown in tick cells infected with the closely-related but less pathogenic flavivirus Langat virus (LGTV)., Results: Differential expression analysis using DESeq resulted in totals of 43 and 83 statistically significantly differentially-expressed transcripts in IDE8 and IRE/CTVM19 cells, respectively. Mass spectrometry detected 76 and 129 statistically significantly differentially-represented proteins in IDE8 and IRE/CTVM19 cells, respectively. Differentially-expressed transcripts and differentially-represented proteins included some that may be involved in innate immune and cell stress responses. Knockdown of the heat-shock proteins HSP90, HSP70 and gp96, the complement-associated protein Factor H and the protease trypsin resulted in increased LGTV replication and production in at least one tick cell line, indicating a possible antiviral role for these proteins. Knockdown of RNAi-associated proteins Argonaute and Dicer, which were included as positive controls, also resulted in increased LGTV replication and production in both cell lines, confirming their role in the antiviral RNAi pathway., Conclusions: This systems biology approach identified several molecules that may be involved in the tick cell innate immune response against flaviviruses and highlighted that ticks, in common with other invertebrate species, have other antiviral responses in addition to RNAi.

Ticks (Acari: Ixodida) are arthropod ectoparasites dependent on a bloodmeal from a vertebrate host at each developmental stage for completion of their life cycle. This tick feeding cycle impacts animal health by causing damage to hides, secondary infections, immune reactions and diseases caused by transmission of pathogens. The genus Ixodes includes several medically important species that vector diseases, including granulocytic anaplasmosis and Lyme disease. I. scapularis, commonly called the black-legged or deer tick, is a medically-important tick species in North America and therefore was the first tick genome to be sequenced, thus serving as an important resource for tick research. This Primer focuses on the normal developmental cycle and laboratory rearing of I. scapularis. Definition of normal morphology, along with a consistent source of laboratory-reared I. scapularis, are fundamental for all aspects of future research, especially the effects of genetic manipulation and the evaluation of tick vaccine efficacy. Recent research important for the advancement of tick research, namely the development of tick cell culture systems for study of ticks and tick-borne pathogens, RNA interference for genetic manipulation of ticks and discovery of candidate antigens for development of tick vaccines, are briefly presented along with areas to target for future research.

Background: Phylogeographical studies allow precise genetic comparison of specimens, which were collected over large geographical ranges and belong to the same or closely related animal species. These methods have also been used to compare ticks of veterinary-medical importance. However, relevant data are missing in the case of ixodid ticks of bats, despite (1) the vast geographical range of both Ixodes vespertilionis and Ixodes simplex, and (2) the considerable uncertainty in their taxonomy, which is currently unresolvable by morphological clues., Methods: In the present study 21 ticks were selected from collections or were freshly removed from bats or cave walls in six European and four Asian countries. The DNA was extracted and PCRs were performed to amplify part of the cytochrome oxidase I (COI), 16S and 12S rDNA genes, followed by sequencing for identification and molecular-phylogenetic comparison., Results: No morphological differences were observed between Ixodes vespertilionis specimens from Spain and from other parts of Europe, but corresponding genotypes had only 94.6 % COI sequence identity. An I. vespertilionis specimen collected in Vietnam was different both morphologically and genetically (i.e. with only 84.1 % COI sequence identity in comparison with I. vespertilionis from Europe). Two ticks (collected in Vietnam and in Japan) formed a monophyletic clade and shared morphological features with I. ariadnae, recently described and hitherto only reported in Europe. In addition, two Asiatic specimens of I. simplex were shown to differ markedly from European genotypes of the same species. Phylogenetic relationships of ticks showed similar clustering patterns with those of their associated bat host species., Conclusions: Although all three ixodid bat tick species evaluated in the present study appear to be widespread in Eurasia, they exhibit pronounced genetic differences. Data of this study also reflect that I. vespertilionis may represent a species complex.

Anaplasma phagocytophilum is an emerging zoonotic pathogen transmitted by Ixodes scapularis that causes human granulocytic anaplasmosis. Here, a high throughput quantitative proteomics approach was used to characterize A. phagocytophilum proteome during rickettsial multiplication and identify proteins involved in infection of the tick vector, I. scapularis. The first step in this research was focused on tick cells infected with A. phagocytophilum and sampled at two time points containing 10-15% and 65-71% infected cells, respectively to identify key bacterial proteins over-represented in high percentage infected cells. The second step was focused on adult female tick guts and salivary glands infected with A. phagocytophilum to compare in vitro results with those occurring during bacterial infection in vivo. The results showed differences in the proteome of A. phagocytophilum in infected ticks with higher impact on protein synthesis and processing than on bacterial replication in tick salivary glands. These results correlated well with the developmental cycle of A. phagocytophilum, in which cells convert from an intracellular reticulated, replicative form to the nondividing infectious dense-core form. The analysis of A. phagocytophilum differentially represented proteins identified stress response (GroEL, HSP70) and surface (MSP4) proteins that were over-represented in high percentage infected tick cells and salivary glands when compared to low percentage infected cells and guts, respectively. The results demonstrated that MSP4, GroEL and HSP70 interact and bind to tick cells, thus playing a role in rickettsia-tick interactions. The most important finding of these studies is the increase in the level of certain bacterial stress response and surface proteins in A. phagocytophilum-infected tick cells and salivary glands with functional implication in tick-pathogen interactions. These results gave a new dimension to the role of these stress response and surface proteins during A. phagocytophilum infection in ticks. Characterization of Anaplasma proteome contributes information on host-pathogen interactions and provides targets for development of novel control strategies for pathogen infection and transmission.

Anaplasma phagocytophilum is an intracellular rickettsial pathogen transmitted by Ixodes spp. ticks, which causes granulocytic anaplasmosis in humans, horses and dogs and tick-borne fever (TBF) in ruminants. In the United States, human granulocytic anaplasmosis (HGA) is highly prevalent while TBF has not been reported. However, in Europe the situation is the opposite, with high prevalence for TBF in sheep and low prevalence of HGA. The origin of these differences has not been identified and our hypothesis is that different A. phagocytophilum isolates impact differently on tick vector capacity through inhibition of apoptosis to establish infection of the tick vector. In this study we used three different isolates of A. phagocytophilum of human, canine and ovine origin to infect the Ixodes ricinus-derived cell line IRE/CTVM20 and the Ixodes scapularis-derived cell line ISE6 in order to characterize the effect of infection on the level of tick cell apoptosis. Inhibition of apoptosis was observed by flow cytometry as early as 24h post-infection for both tick cell lines and all three isolates of A. phagocytophilum, suggesting that pathogen infection inhibits apoptotic pathways to facilitate infection independently of the origin of the A. phagocytophilum isolate and tick vector species. However, infection with A. phagocytophilum isolates inhibited the intrinsic apoptosis pathway at different levels in I. scapularis and I. ricinus cells. These results suggested an impact of vector-pathogen co-evolution on the adaptation of A. phagocytophilum isolates to grow in tick cells as each isolate grew better in the tick cell line derived from its natural vector species. These results increase our understanding of the mechanisms of A. phagocytophilum infection and multiplication and suggest that multiple mechanisms may affect disease prevalence in different geographical regions., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Tudor staphylococcal nuclease (Tudor-SN) and Argonaute (Ago) are conserved components of the basic RNA interference (RNAi) machinery with a variety of functions including immune response and gene regulation. The RNAi machinery has been characterized in tick vectors of human and animal diseases but information is not available on the role of Tudor-SN in tick RNAi and other cellular processes. Our hypothesis is that tick Tudor-SN is part of the RNAi machinery and may be involved in innate immune response and other cellular processes. To address this hypothesis, Ixodes scapularis and I. ricinus ticks and/or cell lines were used to annotate and characterize the role of Tudor-SN in dsRNA-mediated RNAi, immune response to infection with the rickettsia Anaplasma phagocytophilum and the flaviviruses TBEV or LGTV and tick feeding. The results showed that Tudor-SN is conserved in ticks and involved in dsRNA-mediated RNAi and tick feeding but not in defense against infection with the examined viral and rickettsial pathogens. The effect of Tudor-SN gene knockdown on tick feeding could be due to down-regulation of genes that are required for protein processing and blood digestion through a mechanism that may involve selective degradation of dsRNAs enriched in G:U pairs that form as a result of adenosine-to-inosine RNA editing. These results demonstrated that Tudor-SN plays a role in tick RNAi pathway and feeding but no strong evidence for a role in innate immune responses to pathogen infection was found.

Natural foci of ticks, pathogens, and vertebrate reservoirs display complex relationships that are key to the circulation of pathogens and infection dynamics through the landscape. However, knowledge of the interaction networks involved in transmission of tick-borne pathogens are limited because empirical studies are commonly incomplete or performed at small spatial scales. Here, we applied the methodology of ecological networks to quantify >14,000 interactions among ticks, vertebrates, and pathogens in the western Palearctic. These natural networks are highly structured, modular, coherent, and nested to some degree. We found that the large number of vertebrates in the network contributes to its robustness and persistence. Its structure reduces interspecific competition and allows ample but modular circulation of transmitted pathogens among vertebrates. Accounting for domesticated hosts collapses the network's modular structure, linking groups of hosts that were previously unconnected and increasing the circulation of pathogens. This framework indicates that ticks and vertebrates interact along the shared environmental gradient, while pathogens are linked to groups of phylogenetically close reservoirs.

Background: Recently a new hard tick species, Ixodes ariadnae has been discovered, adding to the two known ixodid tick species (I. vespertilionis and I. simplex) of bats in Europe., Findings: Scanning electron microscopic comparison of adult females of these species shows morphological differences concerning the palps, the scutum, the Haller's organ, the coxae, as well as the arrangement and fine structure of setae. Molecular analysis of 10 geographically different isolates revealed 90-95% sequence homology in the 12S and 16S rDNA genes of bat tick species. Based on 12S rDNA sequences, genotypes of I. ariadnae clustered closest to I. simplex, whereas according to their 16S rDNA gene they were closest to I. vespertilionis. The subolesin gene of I. ariadnae had only 91% sequence homology with that of I. ricinus, and is the longest known among hard tick species., Conclusions: The present study illustrates the morphology and clarifies the phylogenetic relationships of the three known bat tick species that occur in Europe. According to its subolesin gene I. ariadnae may have a long evolutionary history.

Anaplasma phagocytophilum, transmitted by ticks of the genus Ixodes, was first described in Scotland as the agent of tick-borne fever in sheep and more recently as the cause of human granulocytic anaplasmosis in the U.S. and Europe. We previously reported sheep as an experimental host for the human NY-18 isolate of A. phagocytophilum. While clinical signs were not observed and infected granulocytes were not seen in stained blood smears, these sheep served as a good host for infection of ticks. In this research we characterized tick feeding sites to better understand tick/host/pathogen interactions. Ixodes scapularis adults were allowed to feed for 2 and 4 days on experimentally infected sheep, after which biopsies were taken beneath tick feeding sites for histopathology, PCR and immunohistochemistry (IHC) studies. In addition, the expression of selected immune response genes was studied in blood and feeding site biopsies. While necrosis was too advanced in 4-day biopsies for accurate cell counts, higher numbers of eosinophils and neutrophils were found in 2-day biopsies from infected sheep as compared with the uninfected controls. An unexpected result was the documentation of higher dermal inflammation in infected sheep at sites without ticks. A. phagocytophilum infected granulocytes were localized by immunohistochemistry (IHC) in skin biopsies using rabbit antibodies against the recombinant A. phagocytophilum major surface protein 4 as the primary antibody for indirect peroxidase-anti-peroxidase and fluorescent antibody IHC. These infected cells are likely to be the source of infection for ticks. Sheep therefore served as good hosts for studying host/pathogen/tick interactions of this human strain of A. phagocytophilum, and provided a means of producing infected ticks for future studies on tick/pathogen developmental and transmission cycles., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

This article documents the public availability of a global transcriptome comparison between Lyme disease tick vectors, Ixodes scapularis and Ixodes ricinus., (© 2014 John Wiley & Sons Ltd.)

The hard-bodied tick Ixodes ricinus (castor bean tick) is the most common tick species in Europe. I. ricinus is a vector of the causative agents of diseases that affect humans and animals including tick-borne encephalitis, borreliosis, tick-borne fever and babesiosis. The innate immune system provides ticks with quite an efficient defence against some pathogenic microorganisms in the event of their penetration into the tick body or through the blood meal. Antimicrobial peptides (AMPs) constitute an important feature of the tick immune system. Defensins are a well-known class of AMPs. Members of the defensin family of proteins have been reported in several tick species. So far, only two defensins had been identified from I. ricinus. In this study, we report the identification of six novel putative defensins from I. ricinus at the genomic and transcriptional levels. At the genomic level they show differences with one being intronless, while others contain two introns. The expression pattern of these molecules in the salivary glands, midgut, ovary, Malpighian tubules, haemolymph and the tick cell line IRE/CTVM19 was determined. Some of them are tissue specific while others seem to be ubiquitous. Molecular and phylogenetic analyses show that these novel members of the I. ricinus defensin family differ phylogenetically and structurally; nevertheless, the cysteine pattern is highly conserved among the family members. Finally, antimicrobial-peptide prediction tools were used to predict putative antimicrobial activity of our defensins. They show putative antimicrobial activity mainly against Gram-positive bacteria. This study displays the diversity of the defensin family in the tick I. ricinus., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Anaplasma phagocytophilum causes human granulocytic anaplasmosis. Infection with this zoonotic pathogen affects gene expression in both the vertebrate host and the tick vector, Ixodes scapularis. Here, we identified new genes, including spectrin alpha chain or alpha-fodrin (CG8) and voltage-dependent anion-selective channel or mitochondrial porin (T2), that are involved in A. phagocytophilum infection/multiplication and the tick cell response to infection. The pathogen downregulated the expression of CG8 in tick salivary glands and T2 in both the gut and salivary glands to inhibit apoptosis as a mechanism to subvert host cell defenses and increase infection. In the gut, the tick response to infection through CG8 upregulation was used by the pathogen to increase infection due to the cytoskeleton rearrangement that is required for pathogen infection. These results increase our understanding of the role of tick genes during A. phagocytophilum infection and multiplication and demonstrate that the pathogen uses similar strategies to establish infection in both vertebrate and invertebrate hosts.

Background: Tick Subolesin and its ortholog in insects and vertebrates, Akirin, have been suggested to play a role in the immune response through regulation of nuclear factor-kappa B (NF-kB)-dependent and independent gene expression via interaction with intermediate proteins that interact with NF-kB and other regulatory proteins, bind DNA or remodel chromatin to regulate gene expression. The objective of this study was to characterize the structure and regulation of subolesin in Ixodes scapularis. I. scapularis is a vector of emerging pathogens such as Borrelia burgdorferi, Anaplasma phagocytophilum and Babesia microti that cause in humans Lyme disease, anaplasmosis and babesiosis, respectively. The genome of I. scapularis was recently sequenced, and this tick serves as a model organism for the study of vector-host-pathogen interactions. However, basic biological questions such as gene organization and regulation are largely unknown in ticks and other arthropod vectors., Principal Findings: The results presented here provide evidence that subolesin/akirin are evolutionarily conserved at several levels (primary sequence, gene organization and function), thus supporting their crucial biological function in metazoans. These results showed that NF-kB (Relish) is involved in the regulation of subolesin expression in ticks, suggesting that as in other organisms, different NF-kB integral subunits and/or unknown interacting proteins regulate the specificity of the NF-kB-mediated gene expression. These results suggested a regulatory network involving cross-regulation between NF-kB (Relish) and Subolesin and Subolesin auto-regulation with possible implications in tick immune response to bacterial infection., Significance: These results advance our understanding of gene organization and regulation in I. scapularis and have important implications for arthropod vectors genetics and immunology highlighting the possible role of NF-kB and Subolesin/Akirin in vector-pathogen interactions and for designing new strategies for the control of vector infestations and pathogen transmission.

The importance of Dermacentor spp. in the transmission of tick-borne pathogens is not well recognized in Europe. To investigate the role of Dermacentor spp. in the transmission of tick-borne pathogens, questing ticks were collected in 9 sites from southern to northwestern France (Camargue Delta to Eastern Brittany) where Dermacentor spp. exist and tick-borne diseases had occurred previously. Three tick species were collected during the spring and autumn of 2009. Collected ticks (both males and females) included D. marginatus (n=377), D. reticulatus (n=74), and I. ricinus (n=45). All ticks were analyzed by PCR or reverse line blot for the presence of pathogens' DNA. Pathogens analyzed were based on veterinarian reports and included Anaplasma phagocytophilum, Coxiella burnetii, Anaplasma marginale, Borrelia burgdorferi, Bartonella spp., Babesia spp., Theileria spp., and Francisella sp. Francisella tularensis was not detected in any of the analyzed ticks. In D. marginatus, infection prevalence for A. phagocytophilum (3%) was similar to that found in I. ricinus in Europe. Other pathogens present in D. marginatus included A. marginale (0.5%), Bartonella spp. (9%), C. burnetii (12%), F. philomiragia (1.3%), and Theileria annulata/Babesia bovis (0.3%), which were detected for the first time in France. Pathogens detected in D. reticulatus included A. marginale (1%), Bartonella spp. (12%), C. burnetii (16%), Borrelia spp. (1.5%), and F. philomiragia (19%). Pathogens detected in I. ricinus included A. phagocytophilum (41%), Bartonella spp. (9%), C. burnetii (18%), A. marginale (1%), Borrelia spp. (4.5%), and Babesia sp. (7%). This study represents the first epidemiological approach to characterize tick-borne pathogens infecting Dermacentor spp. in France and that may be transmitted by ticks from this genus. Further experiments using experimental infections and transmission may be now conducted to analyze vector competency of Dermacentor spp. for these pathogens and to validate such hypothesis.

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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Anaplasma phagocytophilum, first identified as a pathogen of ruminants in Europe, has more recently been recognized as an emerging tick-borne pathogen of humans in the U.S. and Europe. A. phagocytophilum is transmitted by Ixodes spp., but the tick developmental cycle and pathogen/vector interactions have not been fully described. In this research, we report on the experimental infection of sheep with the human NY-18 isolate of A. phagocytophilum which then served as a host for infection of I. scapularis nymphs and adults. A. phagocytophilum was propagated in the human promyelocytic cell line, HL-60, and the infected cell cultures were then used to infect sheep by intravenous inoculation. Infections in sheep were confirmed by PCR and an Anaplasma-competitive ELISA. Clinical signs were not apparent in any of the infected sheep, and only limited hematologic and mild serum biochemical abnormalities were identified. While A. phagocytophilum morulae were rarely seen in neutrophils, blood film evaluation revealed prominent large granular lymphocytes, occasional plasma cells, and rare macrophages. Upon necropsy, gross lesions were restricted to the lymphoid system. Mild splenomegaly and lymphadenomegaly with microscopic evidence of lymphoid hyperplasia was observed in all infected sheep. Female I. scapularis that were allowed to feed and acquire infection on each of the 3 experimentally infected sheep became infected with A. phagocytophilum as determined by PCR of guts (80-87%) and salivary glands (67-100%). Female I. scapularis that acquired infection as nymphs on an experimentally infected sheep transmitted A. phagocytophilum to a susceptible sheep, thus confirming transstadial transmission. Sheep proved to be a good host for the production of I. scapularis infected with this human isolate of A. phagocytophilum, which can be used as a model for future studies of the tick/pathogen interface., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

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

We characterized the effects of subolesin and heat shock protein (HSP) expression on Ixodes scapularis Say (Acari: Ixodidae) stress responses to heat shock and feeding, questing behaviour and Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae) infection. Ticks and cultured tick cells were analysed before and after subolesin, hsp20 and hsp70 gene knock-down by RNA interference. The results of these studies confirm that HSPs are involved in the tick cell response to heat stress and that subolesin and HSPs are both involved in the tick response to blood-feeding stress and A. phagocytophilum infection. Subolesin and hsp20 are involved in the tick protective response to A. phagocytophilum infection and hsp70 expression may be manipulated by the pathogen to increase infectivity. Importantly, these results demonstrate that subolesin, hsp20 and hsp70 expression also affect tick questing behaviour. Overall, this research demonstrates a relationship between hsp and subolesin expression and tick stress responses to heat shock and blood feeding, A. phagocytophilum infection and questing behaviour, thereby extending our understanding of the tick-host-pathogen interface., (© 2011 The Authors. Medical and Veterinary Entomology © 2011 The Royal Entomological Society.)

Red deer (Cervus elaphus) and white-tailed deer (Odocoileus virginianus) are hosts for different tick species and tick-borne pathogens and play a role in tick dispersal and maintenance in some regions. These factors stress the importance of controlling tick infestations in deer and several methods such as culling and acaricide treatment have been used. Tick vaccines are a cost-effective alternative for tick control that reduced cattle tick infestations and tick-borne pathogens prevalence while reducing the use of acaricides. Our hypothesis is that vaccination with vector protective antigens can be used for the control of tick infestations in deer. Herein, three experiments were conducted to characterize (1) the antibody response in red deer immunized with recombinant BM86, the antigen included in commercial tick vaccines, (2) the antibody response and control of cattle tick infestations in white-tailed deer immunized with recombinant BM86 or tick subolesin (SUB) and experimentally infested with Rhipicephalus (Boophilus) microplus, and (3) the antibody response and control of Hyalomma spp. and Rhipicephalus spp. field tick infestations in red deer immunized with mosquito akirin (AKR), the SUB ortholog and candidate protective antigen against different tick species and other ectoparasites. The results showed that deer produced an antibody response that correlated with the reduction in tick infestations and was similar to other hosts vaccinated previously with these antigens. The overall vaccine efficacy was similar between BM86 (E=76%) and SUB (E=83%) for the control of R. microplus infestations in white-tailed deer. The field trial in red deer showed a 25-33% (18-40% when only infested deer were considered) reduction in tick infestations, 14-20 weeks after the first immunization. These results demonstrated that vaccination with vector protective antigens could be used as an alternative method for the control of tick infestations in deer to reduce tick populations and dispersal in regions where deer are relevant hosts for these ectoparasites., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Four white-tailed deer were inoculated with either the Ap-V1 or NY-18 strain of Anaplasma phagocytophilum. Ixodes scapularis nymphs were then allowed to acquistion feed on the inoculated deer and molt to adults. Only an Ap-V1 infected deer was infected persistently and able to infect nymphal Ixodes scapularis. Molted adult ticks maintained Ap-V1 infection as demonstrated by PCR and microscopy. We report, for the first time, a morphologic description of A. phagocytophilum in I. scapularis.

Bovine anaplasmosis is a tick-borne hemolytic disease of cattle that occurs worldwide caused by the intraerythrocytic rickettsiae Anaplasma marginale. Control measures, including use of acaricides, administration of antibiotics and vaccines, have varied with geographic location. Our research is focused on the tick-pathogen interface for development of new vaccine strategies with the goal of reducing anaplasmosis, tick infestations and the vectorial capacity of ticks. Toward this approach, we have targeted (1) development of an A. marginale cell culture system to provide a non-bovine antigen source, (2) characterization of an A. marginale adhesion protein, and (3) identification of key tick protective antigens for reduction of tick infestations. A cell culture system for propagation of A. marginale was developed and provided a non-bovine source of A. marginale vaccine antigen. The A. marginale adhesion protein, MSP1a, was characterized and use of recombinant MSP1a in vaccine formulations reduced clinical anaplasmosis and infection levels in ticks that acquired infection on immunized cattle. Most recently, we identified a tick-protective antigen, subolesin, that reduced tick infestations, as well as the vectorial capacity of ticks for acquisition and transmission of A marginale. This integrated approach to vaccine development shows promise for developing new strategies for control of bovine anaplasmosis.

Antigens protective against Ixodes scapularis infestations were identified by cDNA expression library immunization (ELI) and analysis of expressed sequenced tags (EST). Three cDNAs protective against larval tick infestations, 4F8, with homology to a nucleotidase, and 4D8 and 4E6 of unknown function, were characterized and obtained as recombinant proteins for immunization studies. Vaccination trials with recombinant proteins demonstrated an effect of these antigens against I. scapularis larvae in a mouse model. Herein, we evaluated the effect of recombinant antigens on I. scapularis adult infestations on immunized sheep. Vaccination with recombinant 4D8, 4F8, 4E6 and the combination of all three antigens reduced adult tick infestations by 58, 12, 20, and 16%, respectively, when compared to the control group but was statistically significant for 4D8 and 4F8 only. Oviposition was reduced by 22-49% in all groups immunized with recombinant tick antigens (P<0.05). The overall efficacy of vaccine formulations considering the effect on tick infestations and oviposition averaged 33-71%. These antigens, and especially 4D8, appear to be good candidates for continued development of a vaccine for control of tick infestations.