Vaccines that target blood-feeding disease vectors, such as mosquitoes and ticks, have the potential to protect against the many diseases caused by vector-borne pathogens. We tested the ability of an anti-tick vaccine derived from a tick cement protein (64TRP) of Rhipicephalus appendiculatus to protect mice against tick-borne encephalitis virus (TBEV) transmitted by infected Ixodes ricinus ticks. The vaccine has a “dual action” in immunized animals: when infested with ticks, the inflammatory and immune responses first disrupt the skin feeding site, resulting in impaired blood feeding, and then specific anti-64TRP antibodies cross-react with midgut antigenic epitopes, causing rupture of the tick midgut and death of engorged ticks. Three parameters were measured: “transmission,” number of uninfected nymphal ticks that became infected when cofeeding with an infected adult female tick; “support,” number of mice supporting virus transmission from the infected tick to cofeeding uninfected nymphs; and “survival,” number of mice that survived infection by tick bite and subsequent challenge by intraperitoneal inoculation of a lethal dose of TBEV. We show that one dose of the 64TRP vaccine protects mice against lethal challenge by infected ticks; control animals developed a fatal viral encephalitis. The protective effect of the 64TRP vaccine was comparable to that of a single dose of a commercial TBEV vaccine, while the transmission-blocking effect of 64TRP was better than that of the antiviral vaccine in reducing the number of animals supporting virus transmission. By contrast, the commercial antitick vaccine (TickGARD) that targets only the tick's midgut showed transmission-blocking activity but was not protective. The 64TRP vaccine demonstrates the potential to control vector-borne disease by interfering with pathogen transmission, apparently by mediating a local cutaneous inflammatory immune response at the tick-feeding site.
Blood-sucking vectors such as mosquitoes and ticks transmit hundreds of micro-organisms that cause diseases like malaria and Lyme disease. Controlling so many diseases is an enormous challenge. A new idea is to make vaccines against the vectors rather than against all the individual disease agents they carry. The authors examined this hypothesis using a vaccine prepared from tick cement. This cement is secreted by ticks to help them attach to a human or animal to feed. A mouse model was used in which mice were infested with ticks infected with tick-borne encephalitis virus (TBEV), the most important vector-borne virus in Europe and northern Asia. The control mice developed fatal encephalitis and died about a week after being bitten by the infected tick. By contrast, the tick cement vaccine gave protection similar to the level seen in mice immunized with a single shot of the commercial TBEV vaccine for humans. However, a commercial tick vaccine used to control cattle ticks did not protect the mice. The authors' tick cement vaccine appeared to work by causing a cellular immune response in the skin where ticks were feeding. These results show that it is feasible to produce a vaccine against a tick that protects against the disease agent it transmits.
Ixodes scapularis, the black-legged tick, vectors several human pathogens including Borrelia burgdorferi, the agent of Lyme disease in North America. Pathogen transmission to the vertebrate host occurs when infected ticks feed on the mammalian host to obtain a blood meal. Efforts to understand how the tick confronts host hemostatic mechanisms and imbibes a fluid blood meal have largely focused on the anticoagulation strategies of tick saliva. The blood meal that enters the tick gut remains in a fluid state for several days during the process of feeding, and the role of the tick gut in maintaining the blood-meal fluid is not understood. We now demonstrate that the tick gut produces a potent inhibitor of thrombin, a key enzyme in the mammalian coagulation cascade. Chromatographic fractionation of engorged tick gut proteins identified one predominant thrombin inhibitory activity associated with an approximately 18 kDa protein, henceforth referred to as Ixophilin. The ixophilin gene was preferentially transcribed in the guts of feeding nymphs. Expression began after 24 hours of feeding, coincident with the flow of host blood into the tick gut. Immunity against Ixophilin delayed tick feeding, and decreased feeding efficiency significantly. Surprisingly, immunity against Ixophilin resulted in increased Borrelia burgdorferi transmission to the host, possibly due to delayed feeding and increased transmission opportunity. These observations illuminate the potential drawbacks of targeting individual tick proteins in a functional suite. They also underscore the need to identify the “anticoagulome” of the tick gut, and to prioritize a critical subset of anticoagulants that could be targeted to efficiently thwart tick feeding, and block pathogen transmission to the vertebrate host.
Borrelia burgdorferi, the causative agent of Lyme disease, is transmitted to humans by bite of Ixodes scapularis ticks. The mechanisms by which the bacterium is transmitted from vector to host are poorly understood. In this study, we show that the F(ab)2 fragments of BBE31, a B.burgdorferi outer-surface lipoprotein, interfere with the migration of the spirochete from tick gut into the hemolymph during tick feeding. The decreased hemolymph infection results in lower salivary glands infection, and consequently attenuates mouse infection by tick-transmitted B. burgdorferi. Using a yeast surface display approach, a tick gut protein named TRE31 was identified to interact with BBE31. Silencing tre31 also decreased the B. burgdorferi burden in the tick hemolymph. Delineating the specific spirochete and arthropod ligands required for B. burgdorferi movement in the tick may lead to new strategies to interrupt the life cycle of the Lyme disease agent.
Lyme disease, the most common tick-borne illness in North America, is caused by Borrelia burgdorferi. Currently, spirochete and tick molecules that facilitate Borrelia migration within the vector, a key step for mammalian infection by tick-transmitted spirochetes, have not yet been identified. In this study, we show that F(ab)2 fragments of BBE31, a B.burgdorferi outer-surface lipoprotein, interfere with the spirochete migration from the tick gut into the hemolymph. Our results indicated that decreased hemolymph infection by blocking BBE31 resulted in lower salivary glands infection, which eventually attenuated murine infection by tick-transmitted B.burgdorferi. We also found that a tick gut protein TRE31 enables Borrelia movement by interacting with BBE31. This finding provides novel insights into the transmission of spirochete within the vector and provides potential vaccine targets to block the microbial life cycle within the vector.
Survival of Borrelia burgdorferi in ticks and mammals is facilitated, at least in part, by the selective expression of lipoproteins. Outer surface protein (Osp) A participates in spirochete adherence to the tick gut. As ospB is expressed on a bicistronic operon with ospA, we have now investigated the role of OspB by generating an OspB-deficient B. burgdorferi and examining its phenotype throughout the spirochete life cycle. Similar to wild-type isolates, the OspB-deficient B. burgdorferi were able to readily infect and persist in mice. OspB-deficient B. burgdorferi were capable of migrating to the feeding ticks but had an impaired ability to adhere to the tick gut and survive within the vector. Furthermore, the OspB-deficient B. burgdorferi bound poorly to tick gut extracts. The complementation of the OspB-deficient spirochete in trans, with a wild-type copy of ospB gene, restored its ability to bind tick gut. Taken together, these data suggest that OspB has an important role within Ixodes scapularis and that B. burgdorferi relies upon multiple genes to efficiently persist in ticks.
Lyme disease is the most common vector-borne disease in North America and Europe. The causative agent Borrelia burgdorferi is a bacterium that is maintained in an enzoonotic cycle between Ixodes ticks and a large range of mammals. Accidental encounters of infected Ixodes ticks with humans results in the transmission of B. burgdorferi and subsequent Lyme disease. Given that global control efforts have met with limited success, the need for developing novel interventions to combat this infection has become all the more vital. A better understanding of how B. burgdorferi interacts with its vector might lead to new ideas for combating the Lyme disease. B. burgdorferi upregulates outer surface protein (Osp) A and B during entry into ticks, and OspA contributes to the colonization of bacterium within the vector gut. We now demonstrate that OspB also facilitates the colonization and survival of B. burgdorferi in ticks. This work provides the basis for future studies as to how this protein facilitates interaction of B. burgdorferi to the tick gut and thus ultimately a basis for the development of novel strategies to interrupt the spirochete life cycle.
The agents of Lyme disease (Borrelia burgdorferi) and human granulocytic ehrlichiosis (Ehrlichia phagocytophila) are both transmitted by the tick Ixodes scapularis. In nature, ticks are often infected with both agents simultaneously. We studied whether previous infection with either Borrelia or Ehrlichia in ticks would affect acquisition and transmission of a second pathogen. Ehrlichia-infected I. scapularis nymphs were fed upon Borrelia-infected mice, and Borrelia-infected I. scapularis nymphs were fed upon Ehrlichia-infected mice. The efficiency with which previously infected nymphal ticks acquired a second pathogen from infected hosts was compared to that of uninfected ticks. An average of 51% ± 15% of ticks acquired Ehrlichia from infected mice regardless of their prior infection status with Borrelia. An average of 85% ± 10% of ticks acquired Borrelia from infected mice regardless of their prior infection status with Ehrlichia. Also, we assessed the efficiency with which individual nymphs could transmit either agent alone, or both agents simultaneously, to individual susceptible hosts. An average of 76% ± 9% of Borrelia-infected ticks and 84% ± 10% of Ehrlichia-infected ticks transmitted these agents to mice regardless of the presence of the other pathogen. There was no evidence of interaction between the agents of Lyme disease and human granulocytic ehrlichiosis in I. scapularis ticks. The presence of either agent in the ticks did not affect acquisition of the other agent from an infected host. Transmission of the agents of Lyme disease and human granulocytic ehrlichiosis by individual ticks was equally efficient and independent. Dually infected ticks transmitted each pathogen to susceptible hosts as efficiently as ticks infected with only one pathogen.
Ixodid ticks are vectors of human diseases such as Lyme disease, babesiosis, anaplasmosis, and tick-borne encephalitis. These diseases cause significant morbidity and mortality worldwide and are transmitted to humans during tick feeding. The tick-host-pathogen interface is a complex environment where host responses are modulated by the molecules in tick saliva to enable the acquisition of a blood meal. Disruption of host responses at the site of the tick bite may also provide an advantage for pathogens to survive and replicate. Thus, the molecules in tick saliva not only aid the tick in securing a nutrient-rich blood meal, but can also enhance the transmission and acquisition of pathogens. To investigate the effect of feeding and flavivirus infection on the salivary gland transcript expression profile in ticks, a first-generation microarray was developed using ESTs from a cDNA library derived from Ixodes scapularis salivary glands. When the salivary gland transcript profile in ticks feeding over the course of 3 days was compared to that in unfed ticks, a dramatic increase in transcripts related to metabolism was observed. Specifically, 578 transcripts were up-regulated compared to 151 down-regulated transcripts in fed ticks. When specific time points post attachment were analyzed, a temporal pattern of gene expression was observed. When Langat virus-infected ticks were compared to mock-infected ticks, transcript expression changes were observed at all 3 days of feeding. Differentially regulated transcripts include putative secreted proteins, lipocalins, Kunitz domain-containing proteins, anti-microbial peptides, and transcripts of unknown function. These studies identify salivary gland transcripts that are differentially regulated during feeding or in the context of flavivirus infection in Ixodes scapularis nymphs, a medically important disease vector. Further analysis of these transcripts may identify salivary factors that affect the transmission or replication of tick-borne flaviviruses.
Tick vector; Ixodes scapularis; Nymph; Salivary gland; Gene expression; Feeding; Flavivirus
The basic reproduction number of a pathogen, R0, determines whether a pathogen will spread (R0 > 1), when introduced into a fully susceptible population or fade out (R0 < 1), because infected hosts do not, on average, replace themselves. In this paper we develop a simple mechanistic model for the basic reproduction number for a group of tick-borne pathogens that wholly, or almost wholly, depend on horizontal transmission to and from vertebrate hosts. This group includes the causative agent of Lyme disease, Borrelia burgdorferi, and the causative agent of human babesiosis, Babesia microti, for which transmission between co-feeding ticks and vertical transmission from adult female ticks are both negligible. The model has only 19 parameters, all of which have a clear biological interpretation and can be estimated from laboratory or field data. The model takes into account the transmission efficiency from the vertebrate host as a function of the days since infection, in part because of the potential for this dynamic to interact with tick phenology, which is also included in the model. This sets the model apart from previous, similar models for R0 for tick-borne pathogens. We then define parameter ranges for the 19 parameters using estimates from the literature, as well as laboratory and field data, and perform a global sensitivity analysis of the model. This enables us to rank the importance of the parameters in terms of their contribution to the observed variation in R0. We conclude that the transmission efficiency from the vertebrate host to Ixodes scapularis ticks, the survival rate of Ixodes scapularis from fed larva to feeding nymph, and the fraction of nymphs finding a competent host, are the most influential factors for R0. This contrasts with other vector borne pathogens where it is usually the abundance of the vector or host, or the vector-to-host ratio, that determine conditions for emergence. These results are a step towards a better understanding of the geographical expansion of currently emerging horizontally-transmitted tick-borne pathogens such as Babesia microti, as well as providing a firmer scientific basis for targeted use of acaricide or the application of wildlife vaccines that are currently in development.
Basic reproduction number; Global sensitivity analysis; Lyme disease; Ixodes scapularis
Ixodes ricinus is the most widespread and abundant tick in Europe, frequently bites humans, and is the vector of several pathogens including those responsible for Lyme disease, Tick-Borne Encephalitis, anaplasmosis, babesiosis and bartonellosis. These tick-borne pathogens are transmitted to vertebrate hosts via tick saliva during blood feeding, and tick salivary gland (SG) factors are likely implicated in transmission. In order to identify such tick factors, we characterized the transcriptome of female I. ricinus SGs using next generation sequencing techniques, and compared transcriptomes between Bartonella henselae-infected and non-infected ticks. High-throughput sequencing of I. ricinus SG transcriptomes led to the generation of 24,539 isotigs. Among them, 829 and 517 transcripts were either significantly up- or down-regulated respectively, in response to bacterial infection. Searches based on sequence identity showed that among the differentially expressed transcripts, 161 transcripts corresponded to nine groups of previously annotated tick SG gene families, while the others corresponded to genes of unknown function. Expression patterns of five selected genes belonging to the BPTI/Kunitz family of serine protease inhibitors, the tick salivary peptide group 1 protein, the salp15 super-family, and the arthropod defensin family, were validated by qRT-PCR. IrSPI, a member of the BPTI/Kunitz family of serine protease inhibitors, showed the highest up-regulation in SGs in response to Bartonella infection. IrSPI silencing impaired tick feeding, as well as resulted in reduced bacterial load in tick SGs. This study provides a comprehensive analysis of I. ricinus SG transcriptome and contributes significant genomic information about this important disease vector. This in-depth knowledge will enable a better understanding of the molecular interactions between ticks and tick-borne pathogens, and identifies IrSPI, a candidate to study now in detail to estimate its potentialities as vaccine against the ticks and the pathogens they transmit.
I. ricinus is the most common tick species in Europe and the vector for several pathogens, including bacteria from the Bartonella genus. The mechanisms by which ticks modulate their gene expression in response to pathogen infection are poorly understood. In this report, we compared the differential expression of genes expressed in tick salivary glands during B. henselae infection using next generation sequencing techniques. This approach identified 829 and 517 transcripts either significantly up- or down-regulated respectively, in response to bacterial infection. Among them, 161 transcripts corresponded to nine gene family groups previously described in ticks. By silencing the most up-regulated transcript (IrSPI), we demonstrated its implication in both tick feeding and bacterial infection of tick salivary glands. This study demonstrates that a molecular dialogue exists between pathogens and their arthropod vectors and provides, with IrSPI, a candidate to study now in detail to estimate its potentialities as vaccine against the ticks and the pathogens they transmit.
Borrelia burgdorferi transmission to the vertebrate host commences with growth of the spirochete in the tick gut and migration from the gut to the salivary glands. This complex process, involving intimate interactions of the spirochete with the gut epithelium, is pivotal to transmission. We utilized a yeast surface display library of tick gut proteins to perform a global screen for tick gut proteins that might interact with Borrelia membrane proteins. A putative fibronectin type III domain-containing tick gut protein (Ixofin3D) was most frequently identified from this screen and prioritized for further analysis. Immunization against Ixofin3D and RNA interference-mediated reduction in expression of Ixofin3D resulted in decreased spirochete burden in tick salivary glands and in the murine host. Microscopic examination showed decreased aggregation of spirochetes on the gut epithelium concomitant with reduced expression of Ixofin3D. Our observations suggest that the interaction between Borrelia and Ixofin3D facilitates spirochete congregation to the gut during transmission, and provides a “molecular exit” direction for spirochete egress from the gut.
Lyme borreliosis, the most common vector-borne illness in Northeastern parts of USA, is caused by Borrelia burgdorferi sensu lato spirochetes, and transmitted by the Ixodes scapularis ticks. Currently there is no vaccine available to prevent Lyme borreliosis. A better understanding of tick proteins that interact with Borrelia to facilitate spirochete transmission could identify new targets for the development of a tick-based vaccine to prevent Lyme borreliosis. Spirochete growth and exit from the gut is central to transmission, and might involve intimate interactions between the spirochete and the tick gut. We therefore performed a global screen to identify Borrelia-interacting tick gut proteins. One of the four Borrelia-interacting tick proteins, referred to as Ixofin3D, was further characterized. RNA-interference-mediated down-regulation of Ixofin3D resulted in decreased spirochete numbers in the salivary glands and consequently decreased transmission to the host during tick feeding. We demonstrate that Ixofin3D aids spirochete congregation to the gut epithelium, a critical first step that might direct spirochete exit from the gut.
Borrelia burgdorferi, the spirochetal agent of Lyme disease, is transmitted by Ixodes ticks. A vaccine based on B. burgdorferi outer surface protein (Osp) A protects mice from spirochete infection. Here we report on the expression of OspA on spirochetes inside engorging ticks and relate OspA expression to antispirochetal immunity. Spirochetes in the gut of unfed nymphal ticks were stained by an OspA antibody, whereas in feeding ticks, the majority of spirochetes in the gut and salivary glands did not stain with the antibody. Thus, OspA was not expressed on most spirochetes during transmission from the vector to the vertebrate host. To examine the mechanism of protection afforded by OspA antibody, mice were passively immunized with OspA antibody at different times relative to tick attachment. When OspA antibody was administered to mice before or at the time of tick attachment, spirochetal development events in the vector, such as growth and salivary gland invasion, were blocked and the mice were protected from B. burgdorferi infection. When OspA antibody was administered to mice 48 h after tick attachment, spirochetes persisted in the nymphs and the mice were not protected despite the presence of circulating antibodies in the host as well as in the tick blood meal. Thus, OspA immunity appears to be effective only during a narrow window time at the beginning of the blood meal when antibodies bind to OspA-expressing spirochetes in the tick gut and block transmission from the vector to the host.
•Tick saliva proteins Salp15 and Iric-1 promote tick feeding and pathogen transmission.•We established the first bacterial expression system for soluble Salp15 and Iric-1.•Using this system we mapped monoclonal antibody epitopes on Salp15 and Iric-1.•We defined the interaction sites with Borrelia outer surface protein C (OspC).•We elucidated first secondary structure features in Iric-1 by NMR.
Ticks transmit numerous pathogens, including borreliae, which cause Lyme disease. Tick saliva contains a complex mix of anti-host defense factors, including the immunosuppressive cysteine-rich secretory glycoprotein Salp15 from Ixodes scapularis ticks and orthologs like Iric-1 from Ixodesricinus. All tick-borne microbes benefit from the immunosuppression at the tick bite site; in addition, borreliae exploit the binding of Salp15 to their outer surface protein C (OspC) for enhanced transmission. Hence, Salp15 proteins are attractive targets for anti-tick vaccines that also target borreliae. However, recombinant Salp proteins are not accessible in sufficient quantity for either vaccine manufacturing or for structural characterization. As an alternative to low-yield eukaryotic systems, we investigated cytoplasmic expression in Escherichia coli, even though this would not result in glycosylation. His-tagged Salp15 was efficiently expressed but insoluble. Among the various solubility-enhancing protein tags tested, DsbA was superior, yielding milligram amounts of soluble, monomeric Salp15 and Iric-1 fusions. Easily accessible mutants enabled epitope mapping of two monoclonal antibodies that, importantly, cross-react with glycosylated Salp15, and revealed interaction sites with OspC. Free Salp15 and Iric-1 from protease-cleavable fusions, despite limited solubility, allowed the recording of 1H–15N 2D NMR spectra, suggesting partial folding of the wild-type proteins but not of Cys-free variants. Fusion to the NMR-compatible GB1 domain sufficiently enhanced solubility to reveal first secondary structure elements in 13C/15N double-labeled Iric-1. Together, E. coli expression of appropriately fused Salp15 proteins may be highly valuable for the molecular characterization of the function and eventually the 3D structure of these medically relevant tick proteins.
DsbA, thiol:disulfide interchange protein DsbA; GB1, immunoglobulin binding domain B1 of streptococcal protein G; eGFP, enhanced green fluorescent protein; GST, glutathione-S-transferase; HSQC, heteronuclear single quantum coherence; MBP, maltose-binding protein; OspC, outer surface protein C; Salp15, salivary protein of 15 kDa; Trx, thioredoxin; Lyme borreliosis; Protein NMR; Saliva-assisted transmission; Solubility-enhanced fusion proteins; Tick saliva; Tick saliva antibodies
Ixodes ticks are major vectors for human pathogens, such as Borrelia burgdorferi, the causative agent of Lyme disease. Tick saliva contains immunosuppressive molecules that facilitate tick feeding and B. burgdorferi infection. We here demonstrate, to our knowledge for the first time, that the Ixodes scapularis salivary protein Salp15 inhibits adaptive immune responses by suppressing human dendritic cell (DC) functions. Salp15 inhibits both Toll-like receptor- and B. burgdorferi–induced production of pro-inflammatory cytokines by DCs and DC-induced T cell activation. Salp15 interacts with DC-SIGN on DCs, which results in activation of the serine/threonine kinase Raf-1. Strikingly, Raf-1 activation by Salp15 leads to mitogen-activated protein kinase kinase (MEK)-dependent decrease of IL-6 and TNF-α mRNA stability and impaired nucleosome remodeling at the IL-12p35 promoter. These data demonstrate that Salp15 binding to DC-SIGN triggers a novel Raf-1/MEK-dependent signaling pathway acting at both cytokine transcriptional and post-transcriptional level to modulate Toll-like receptor–induced DC activation, which might be instrumental to tick feeding and B. burgdorferi infection, and an important factor in the pathogenesis of Lyme disease. Insight into the molecular mechanism of immunosuppression by tick salivary proteins might provide innovative strategies to combat Lyme disease and could lead to the development of novel anti-inflammatory or immunosuppressive agents.
Upon attachment of the tick, the host elicits both innate and adaptive immune responses directed against the vector. In turn, ticks have developed countermeasures to withstand and evade host immune responses. In the current paper we demonstrate how a tick salivary protein induces immunosuppression of human dendritic cells and how this could facilitate infection with B. burgdorferi, the causative agent of Lyme disease. Insight into the molecular mechanism of immunosuppression by tick salivary proteins might provide innovative strategies to combat Lyme disease or other tick-borne illnesses and could lead to the development of novel anti-inflammatory or immunosuppressive drugs.
The density of spirochetes in field-collected or experimentally infected ticks is estimated mainly by assays based on microscopy. In this study, a real-time quantitative PCR (qPCR) protocol targeting the Borrelia burgdorferi-specific recA gene was adapted for use with a Lightcycler for rapid detection and quantification of the Lyme disease spirochete, B. burgdorferi, in field-collected Ixodes scapularis ticks. The sensitivity of qPCR for detection of B. burgdorferi DNA in infected ticks was comparable to that of a well-established nested PCR targeting the 16S-23S rRNA spacer. Of the 498 I. scapularis ticks collected from four northeastern states (Rhode Island, Connecticut, New York, and New Jersey), 91 of 438 (20.7%) nymphal ticks and 15 of 60 (25.0%) adult ticks were positive by qPCR assay. The number of spirochetes in individual ticks varied from 25 to 197,200 with a mean of 1,964 spirochetes per nymphal tick and a mean of 5,351 spirochetes per adult tick. No significant differences were found in the mean numbers of spirochetes counted either in nymphal ticks collected at different locations in these four states (P = 0.23 by one-way analysis of variance test) or in ticks infected with the three distinct ribosomal spacer restriction fragment length polymorphism types of B. burgdorferi (P = 0.39). A high degree of spirochete aggregation among infected ticks (variance-to-mean ratio of 24,877; moment estimate of k = 0.279) was observed. From the frequency distribution data and previously published transmission studies, we estimated that a minimum of 300 organisms may be required in a host-seeking nymphal tick to be able to transmit infection to mice while feeding on mice. These data indicate that real-time qPCR is a reliable approach for simultaneous detection and quantification of B. burgdorferi infection in field-collected ticks and can be used for ecological and epidemiological surveillance of Lyme disease spirochetes.
Ixodes scapularis ticks transmit a number of human pathogens, including the Lyme disease spirochete Borrelia burgdorferi. I. scapularis suppresses host immunity in the skin to promote feeding and systemically skew T-helper (Th)-cell differentiation toward Th2 cells in secondary lymphoid organs. Although components of tick saliva are known to influence Th-cell polarization, the mechanism whereby tick feeding in the skin modulates regional and systemic Th-cell responses is unknown. In this study, the role of the epidermal Langerhans cell (LC) subset of skin dendritic cells in tick-mediated Th1/Th2-cell immunomodulation was assessed. Mice deficient in LCs (Langerin-DTA mice) exhibited enhanced lymph node (LN) concanavalin A (ConA)-induced Th1 responses after tick infestation in comparison to results for uninfested Langerin-DTA or wild-type (WT) mice, whereas effects on Th2-cell production of interleukin 4 were more variable. Nonetheless, the altered T-cell response did not impact tick feeding or refeeding. Gamma interferon production by ConA-stimulated LN cells of both WT and LC-deficient mice was enhanced by as much as fourfold after B. burgdorferi-infected-tick feeding, indicating that immunomodulatory effects of tick saliva were not able to attenuate the Th1 immune responses induced by this pathogen. Taken together, these findings show a requirement for LCs in the tick-mediated attenuation of Th1 responses in regional lymph nodes but not in the spleens of mice and show that the presence of a pathogen can overcome the Th1-inhibitory effects of tick feeding on the host.
Ticks are vectors of a wide variety of pathogens causing severe diseases in humans and domestic animals. Intestinal digestion of the host blood is an essential process of tick physiology and also a limiting factor for pathogen transmission since the tick gut represents the primary site for pathogen infection and proliferation. Using the model tick Ixodes ricinus, the European Lyme disease vector, we have previously demonstrated by genetic and biochemical analyses that host blood is degraded in the tick gut by a network of acidic peptidases of the aspartic and cysteine classes.
This study reveals the digestive machinery of the I. ricinus during the course of blood-feeding on the host. The dynamic profiling of concentrations, activities and mRNA expressions of the major digestive enzymes demonstrates that the de novo synthesis of peptidases triggers the dramatic increase of the hemoglobinolytic activity along the feeding period. Overall hemoglobinolysis, as well as the activity of digestive peptidases are negligible at the early stage of feeding, but increase dramatically towards the end of the slow feeding period, reaching maxima in fully fed ticks. This finding contradicts the established opinion that blood digestion is reduced at the end of engorgement. Furthermore, we show that the digestive proteolysis is localized intracellularly throughout the whole duration of feeding.
Results suggest that the egressing proteolytic system in the early stage of feeding and digestion is a potential target for efficient impairment, most likely by blocking its components via antibodies present in the host blood. Therefore, digestive enzymes are promising candidates for development of novel 'anti-tick' vaccines capable of tick control and even transmission of tick-borne pathogens.
Ticks are unique among hematophagous arthropods by continuous attachment to host skin and blood feeding for days; complexity and diversity of biologically active molecules differentially expressed in saliva of tick species; their ability to modulate the host defenses of pain and itch, hemostasis, inflammation, innate and adaptive immunity, and wound healing; and, the diverse array of infectious agents they transmit. All of these interactions occur at the cutaneous interface in a complex sequence of carefully choreographed host defense responses and tick countermeasures resulting in an environment that facilitates successful blood feeding and establishment of tick-borne infectious agents within the host. Here, we examine diverse patterns of tick attachment to host skin, blood feeding mechanisms, salivary gland transcriptomes, bioactive molecules in tick saliva, timing of pathogen transmission, and host responses to tick bite. Ticks engage and modulate cutaneous and systemic immune defenses involving keratinocytes, natural killer cells, dendritic cells, T cell subpopulations (Th1, Th2, Th17, Treg), B cells, neutrophils, mast cells, basophils, endothelial cells, cytokines, chemokines, complement, and extracellular matrix. A framework is proposed that integrates tick induced changes of skin immune effectors with their ability to respond to tick-borne pathogens. Implications of these changes are addressed. What are the consequences of tick modulation of host cutaneous defenses? Does diversity of salivary gland transcriptomes determine differential modulation of host inflammation and immune defenses and therefore, in part, the clades of pathogens effectively transmitted by different tick species? Do ticks create an immunologically modified cutaneous environment that enhances specific pathogen establishment? Can tick saliva molecules be used to develop vaccines that block pathogen transmission?
ticks; tick saliva; immune response; pathogen transmission; tick–host interface; immune modulation; skin
Borrelia burgdorferi, the spirochetal agent of Lyme disease, is transmitted by Ixodes ticks. When an infected nymphal tick feeds on a host, the bacteria increase in number within the tick, after which they invade the tick’s salivary glands and infect the host. Antibodies directed against outer surface protein A (OspA) of B. burgdorferi kill spirochetes within feeding ticks and block transmission to the host. In the studies presented here, passive antibody transfer experiments were carried out to determine the OspA antibody titer required to block transmission to the rodent host. OspA antibody levels were determined by using a competitive enzyme-linked immunosorbent assay that measured antibody binding to a protective epitope defined by monoclonal antibody C3.78. The C3.78 OspA antibody titer (>213 μg/ml) required to eradicate spirochetes from feeding ticks was considerably higher than the titer (>6 μg/ml) required to block transmission to the host. Although spirochetes were not eradicated from ticks at lower antibody levels, the antibodies reduced the number of spirochetes within the feeding ticks and interfered with the ability of spirochetes to induce ospC and invade the salivary glands of the vector. OspA antibodies may directly interfere with the ability of B. burgdorferi to invade the salivary glands of the vector; alternately, OspA antibodies may lower the density of spirochetes within feeding ticks below a critical threshold required for initiating events linked to transmission.
Borrelia burgdorferi, the spirochetal agent of Lyme disease, is a vector-borne pathogen that cycles between a mammalian host and tick vector. This complex life cycle requires that the spirochete modulate its gene expression program to facilitate growth and maintenance in these diverse milieus. B. burgdorferi contains an operon that is predicted to encode proteins that would mediate the uptake and conversion of glycerol to dihydroxyacetone phosphate. Previous studies indicated that expression of the operon is elevated at 23°C and is repressed in the presence of the alternative sigma factor RpoS, suggesting that glycerol utilization may play an important role during the tick phase. This possibility was further explored in the current study by expression analysis and mutagenesis of glpD, a gene predicted to encode glycerol 3-phosphate dehydrogenase. Transcript levels for glpD were significantly lower in mouse joints relative to their levels in ticks. Expression of GlpD protein was repressed in an RpoS-dependent manner during growth of spirochetes within dialysis membrane chambers implanted in rat peritoneal cavities. In medium supplemented with glycerol as the principal carbohydrate, wild-type B. burgdorferi grew to a significantly higher cell density than glpD mutant spirochetes during growth in vitro at 25°C. glpD mutant spirochetes were fully infectious in mice by either needle or tick inoculation. In contrast, glpD mutants grew to significantly lower densities than wild-type B. burgdorferi in nymphal ticks and displayed a replication defect in feeding nymphs. The findings suggest that B. burgdorferi undergoes a switch in carbohydrate utilization during the mammal to tick transition. Further, the results demonstrate that the ability to utilize glycerol as a carbohydrate source for glycolysis during the tick phase of the infectious cycle is critical for maximal B. burgdorferi fitness.
Borrelia burgdorferi is the vector-borne pathogen that causes Lyme disease. It has a complex life cycle that involves growth in a tick vector and a mammalian host — two diverse environments that present B. burgdorferi with alternative carbohydrate sources for support of growth. Previous studies suggested that glycerol may be an important nutrient in the tick vector. Here we show that genes predicted to be involved in glycerol metabolism have significantly elevated expression during all tick stages. Repression of expression in the mammalian host is dependent on the alternative sigma factor, RpoS. A mutant that cannot convert glycerol into dihydroxyacetone phosphate to support glycolysis was able to infect mice. In contrast, the mutant was present at significantly lower levels in nymphal ticks, its replication was delayed during nymphal feeding and longer feeding times were required for transmission from nymph to mouse. The results demonstrate that the ability to utilize glycerol as a carbohydrate source for glycolysis during the tick phase of the infectious cycle is critical for maximal B. burgdorferi fitness.
The Lyme disease spirochete Borrelia burgdorferi senses and responds to environmental cues as it transits between the tick vector and vertebrate host. Failure to properly adapt can block transmission of the spirochete and persistence in either vector or host. We previously identified BBD18, a novel plasmid-encoded protein of B. burgdorferi, as a putative repressor of the host-essential factor OspC. In this study, we investigate the in vivo role of BBD18 as a regulatory protein, using an experimental mouse-tick model system that closely resembles the natural infectious cycle of B. burgdorferi. We show that spirochetes that have been engineered to constitutively produce BBD18 can colonize and persist in ticks but do not infect mice when introduced by either tick bite or needle inoculation. Conversely, spirochetes lacking BBD18 can persistently infect mice but are not acquired by feeding ticks. Through site-directed mutagenesis, we have demonstrated that abrogation of spirochete infection in mice by overexpression of BBD18 occurs only with bbd18 alleles that can suppress OspC synthesis. Finally, we demonstrate that BBD18-mediated regulation does not utilize a previously described ospC operator sequence required by B. burgdorferi for persistence in immunocompetent mice. These data lead us to conclude that BBD18 does not represent the putative repressor utilized by B. burgdorferi for the specific downregulation of OspC in the mammalian host. Rather, we suggest that BBD18 exhibits features more consistent with those of a global regulatory protein whose critical role occurs during spirochete acquisition by feeding ticks.
Lyme disease, caused by Borrelia burgdorferi, is the most common arthropod-borne disease in North America. B. burgdorferi is transmitted to humans and other vertebrate hosts by ticks as they take a blood meal. Transmission between vectors and hosts requires the bacterium to sense changes in the environment and adapt. However, the mechanisms involved in this process are not well understood. By determining how B. burgdorferi cycles between two very different environments, we can potentially establish novel ways to interfere with transmission and limit infection of this vector-borne pathogen. We are studying a regulatory protein called BBD18 that we recently described. We found that too much BBD18 interferes with the spirochete’s ability to establish infection in mice, whereas too little BBD18 appears to prevent colonization in ticks. Our study provides new insight into key elements of the infectious cycle of the Lyme disease spirochete.
Disease risk maps are important tools that help ascertain the likelihood of exposure to specific infectious agents. Understanding how climate change may affect the suitability of habitats for ticks will improve the accuracy of risk maps of tick-borne pathogen transmission in humans and domestic animal populations. Lyme disease (LD) is the most prevalent arthropod borne disease in the US and Europe. The bacterium Borrelia burgdorferi causes LD and it is transmitted to humans and other mammalian hosts through the bite of infected Ixodes ticks. LD risk maps in the transboundary region between the U.S. and Mexico are lacking. Moreover, none of the published studies that evaluated the effect of climate change in the spatial and temporal distribution of I. scapularis have focused on this region.
The area of study included Texas and a portion of northeast Mexico. This area is referred herein as the Texas-Mexico transboundary region. Tick samples were obtained from various vertebrate hosts in the region under study. Ticks identified as I. scapularis were processed to obtain DNA and to determine if they were infected with B. burgdorferi using PCR. A maximum entropy approach (MAXENT) was used to forecast the present and future (2050) distribution of B. burgdorferi-infected I. scapularis in the Texas-Mexico transboundary region by correlating geographic data with climatic variables.
Of the 1235 tick samples collected, 109 were identified as I. scapularis. Infection with B. burgdorferi was detected in 45% of the I. scapularis ticks collected. The model presented here indicates a wide distribution for I. scapularis, with higher probability of occurrence along the Gulf of Mexico coast. Results of the modeling approach applied predict that habitat suitable for the distribution of I. scapularis in the Texas-Mexico transboundary region will remain relatively stable until 2050.
The Texas-Mexico transboundary region appears to be part of a continuum in the pathogenic landscape of LD. Forecasting based on climate trends provides a tool to adapt strategies in the near future to mitigate the impact of LD related to its distribution and risk for transmission to human populations in the Mexico-US transboundary region.
Ixodes scapularis; Borrelia burgdorferi; Transboundary disease; Lyme disease risk map; Climate change
Lyme disease is the most common tick-borne illness in the United States. In this paper we explore the contribution of Ixodes scapularis ticks to the pathogenicity of Borrelia burgdorferi in mice. Previously we demonstrated that an isolate of B. burgdorferi sensu stricto (designated N40), passaged 75 times in vitro (N40-75), was infectious but was no longer able to cause arthritis and carditis in C3H mice. We now show that N40-75 spirochetes can readily colonize I. scapularis and multiply during tick engorgement. Remarkably, tick-transmitted N40-75 spirochetes cause disease in mice. N40-75 spirochetes isolated from these animals also retained their pathogenicity when subsequently administered to mice via syringe inoculation. Array analysis revealed that several genes associated with virulence, including bba25, bba65, bba66, bbj09, and bbk32, had higher expression levels in the tick-passaged N40-75 spirochete. These data suggest that transmission of a high-passage attenuated isolate of B. burgdorferi by the arthropod vector results in the generation of spirochetes that have enhanced pathogenesis in mice.
In the northeastern United States, the Lyme disease agent, Borrelia burgdorferi sensu stricto, is maintained by enzoonotic transmission, cycling between white-footed mice (Peromyscus leucopus) and black-legged ticks (Ixodes scapularis). B. burgdorferi sensu stricto is genetically variable and has been divided into three major genotypes based on 16S-23S ribosomal DNA spacer (RST) analysis. To better understand how genetic differences in B. burgdorferi sensu stricto may influence transmission dynamics in nature, we investigated the interaction between an RST1 and an RST3 strain in a laboratory system with P. leucopus mice and I. scapularis ticks. Two groups of mice were infected with either BL206 (RST1) or B348 (RST3). Two weeks later, experimental mice were challenged with the opposite strain, while control mice were challenged with the same strain as that used for the primary infection. The transmission of BL206 and B348 from infected mice was then determined by xenodiagnosis with uninfected larval ticks at weekly intervals for 42 days. Mice in both experimental groups were permissive for infection with the second strain and were able to transmit both strains to the xenodiagnostic ticks. However, the overall transmission efficiencies of BL206 and B348 were significantly different. BL206 was more efficiently transmitted than B348 to xenodiagnostic ticks. Significantly fewer double infections than expected were detected in xenodiagnostic ticks. The results suggest that some B. burgdorferi sensu stricto strains, such as BL206, may be preferentially maintained in transmission cycles between ticks and white-footed mice. Other strains, such as B348, may be more effectively maintained in different tick-vertebrate transmission cycles.
The common tick Ixodes ricinus is the main vector in Europe of the tick-borne encephalitis virus and of several species of the Borrelia burgdorferi sensu lato complex, which are the etiological agents of Lyme borreliosis. The risk to contract bites of I. ricinus is dependent on many factors including the behaviour of both ticks and people. The tick’s site of attachment on the human body and the duration of tick attachment may be of clinical importance. Data on I. ricinus ticks, which were found attached to the skin of people, were analysed regarding potentially stage-specific differences in location of attachment sites, duration of tick attachment (= feeding duration), seasonal and geographical distribution of tick infestation in relation to age and gender of the tick-infested hosts.
During 2008–2009, 1770 tick-bitten persons from Sweden and the Åland Islands removed 2110 I. ricinus ticks. Participants provided information about the date of tick detection and location on their body of each attached tick. Ticks were identified to species and developmental stage. The feeding duration of each nymph and adult female tick was microscopically estimated based on the scutal and the coxal index.
In 2008, participants were tick-bitten from mid-May to mid-October and in 2009 from early April to early November. The infestation pattern of the nymphs was bimodal whereas that of the adult female ticks was unimodal with a peak in late summer. Tick attachment site on the human body was associated with stage of the tick and gender of the human host. Site of attachment seemed to influence the duration of tick feeding. Overall, 63% of nymphs and adult female ticks were detected and removed more than 24 hours after attachment. Older persons, compared to younger ones, and men, compared to women, removed “their” ticks after a longer period of tick attachment.
The infestation behaviour of the different tick stages concerning where on the host’s body the ticks generally will attach and when such ticks generally will be detected and removed in relation to host age and gender, should be of value for the development of prophylactic methods against tick infestation and to provide relevant advice to people on how to avoid or reduce the risk of tick infestation.
Ixodes ricinus; Tick infestation; Tick bite; Attachment site; Feeding behaviour; Feeding duration; Host-seeking behaviour; Seasonal activity; Sweden; Åland
The spread of tick-borne pathogens represents an important threat to human and animal health in many parts of Eurasia. Here, we analysed a 9-year time series of Ixodes ricinus ticks feeding on Apodemus flavicollis mice (main reservoir-competent host for tick-borne encephalitis, TBE) sampled in Trentino (Northern Italy). The tail of the distribution of the number of ticks per host was fitted by three theoretical distributions: Negative Binomial (NB), Poisson-LogNormal (PoiLN), and Power-Law (PL). The fit with theoretical distributions indicated that the tail of the tick infestation pattern on mice is better described by the PL distribution. Moreover, we found that the tail of the distribution significantly changes with seasonal variations in host abundance. In order to investigate the effect of different tails of tick distribution on the invasion of a non-systemically transmitted pathogen, we simulated the transmission of a TBE-like virus between susceptible and infective ticks using a stochastic model. Model simulations indicated different outcomes of disease spreading when considering different distribution laws of ticks among hosts. Specifically, we found that the epidemic threshold and the prevalence equilibria obtained in epidemiological simulations with PL distribution are a good approximation of those observed in simulations feed by the empirical distribution. Moreover, we also found that the epidemic threshold for disease invasion was lower when considering the seasonal variation of tick aggregation.
Our work analyses a 9-year time series of tick co-feeding patterns on Yellow-necked mice. Our data shows a strong heterogeneity, where most mice are parasitised by a small number of ticks while few host a much larger number. We describe the number of ticks per host by the commonly used Negative Binomial model, by the Poisson-LogNormal model, and we propose the Power Law model as an alternative. In our data, the last model seems to better describe the strong heterogeneity. In order to understand the epidemiological consequences, we use a computational model to reproduce a peculiar way of transmission, observed in some cases in nature, where uninfected ticks acquire an infection by feeding on a host where infected ticks are present, without any remarkable epidemiological involvement of the host itself. In particular, we are interested in determining the conditions leading to pathogen spread. We observe that the effective transmission of this infection in nature is highly dependent on the capability of the implemented model to describe the tick burden. In addition, we also consider seasonal changes in tick aggregation on mice, showing its influence on the spread of the infection.
In North America, the black-legged tick, Ixodes scapularis, an obligate haematophagus arthropod, is a vector of several human pathogens including Borrelia burgdorferi, the Lyme disease agent. In this report, we show that the tick salivary gland transcriptome and proteome is dynamic and changes during the process of engorgement. We demonstrate, using a guinea pig model of I. scapularis feeding and B. burgdorferi transmission, that immunity directed against salivary proteins expressed in the first 24 h of tick attachment — and not later — is sufficient to evoke all the hallmarks of acquired tick-immunity, to thwart tick feeding and also to impair Borrelia transmission. Defining this subset of proteins will promote a mechanistic understanding of novel I. scapularis proteins critical for the initiation of tick feeding and for Borrelia transmission.