Saliva,Salivary Gland,and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) ^1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick ^3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick''s enzootic cycle ^14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva ^9,16-19.As a tick feeds the host typically responds with a strong hemostatic and innate immune response ^11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding ^{3,11,20,21,23}. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens ^3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.     

Ixodes ricinus tick saliva modulates tick-borne encephalitis virus infection of dendritic cells

Tick-borne encephalitis virus is an important human pathogen, naturally delivered into host skin via a tick bite. To examine the effects of the virus on dendritic cell biology, we cultured dendritic cells with two tick-borne encephalitis virus strains of different virulence in the presence of Ixodes ricinus tick saliva. Tick saliva treatment increased proportion of virus-infected cells, led to a decrease in virus-induced TNF-α and IL-6 production and to reduced virus-induced apoptosis. Our data indicate that tick saliva modulate virus-mediated alterations in dendritic cells, thus probably being involved in the early infection process in the host.     

Identification of Four Novel Rhipicephalus annulatus Upregulated Salivary Gland Proteins as Candidate Vaccines

The control of Rhipicephalus annulatus ticks in Egypt and other countries relies principally on the application of acaricides which have many drawbacks. Recently, cattle vaccination against ticks showed a potential unconventional approach to control ticks. As a target, salivary glands contain various proteins that may play specific roles during attachment, feeding and may modulate the immune system of the host. We have performed immunoscreening on expression normalized cDNA library to identify unique R. annulatus proteins from salivary gland (RaSal) that are particularly expressed during engorgement. We also present the cloning and sequencing of four novel cDNAs (RaSal1–4) from salivary glands that are expressed during feeding. RaSal4 shows 13 cysteine amino acid residues forming 6 potential disulfide bonds. We detected the expression level of the four genes during embryogenesis in eggs collected at 6, 12 and 18 days after oviposition. RT-PCR analysis detected these proteins at days 12 and 18 while slight amplification was detected at day 6 for only RaSal2. The expression of these salivary genes may put forward new vaccines to control tick infestations and tick-borne diseases.     

Zecken als Krankheitsüberträger: Was tun bei einem Stich?

Because of its wide host‐range and capacity for transmission of multiple pathogens, Ixodes icinus poses a constant threat of human infection. Borrelia burgdorferi is the most prevalent tick‐borne pathogen affecting humans (Lyme Borreliosis), tick‐borne‐encephalitis (TBE) the most important viral tick‐borne disease in Europe. In natural foci the pathogens circulate between infected small mammals and ticks. Knowing the lifecycle of I.ricinus, their multistrategies for host finding, attachment and blood ingestion, we may understand, what makes the tick such an excellent vector. Instructions for individual behaviour in tick areas to avoid tick contact are given. Since transmission is closely related to the feeding period it is helpful to remove an attached tick as soon as possible. Protection against tick‐borne encephalitis by vaccination is possible.     

Preliminary studies on virus and spirochete accumulation in the cement plug of ixodid ticks

We provide evidence that tick-borne encephalitis virus and Borrelia burgdorferi s.l. are accumulated in the cement plug in the host skin within the first few hours after tick attachment. Extirpation of the tick without the cement plug, even very soon after the attachment, did not prevent the transmission by Ixodes ricinus, Ixodes persulcatus or Dermacentor reticulatus to mice. This was within 1 hour in the case of the TBE virus and after 20–22 h of attachment, in the case of Borrelia and I. persulcatus. The epidemiological significance of these findings is discussed.     

Pattern of Tick Aggregation on Mice: Larger Than Expected Distribution Tail Enhances the Spread of Tick-Borne Pathogens

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.     

Tick modulation of host immunity: an important factor in pathogen transmission.

Immunological interactions at the tick host interface involve innate and specific acquired host immune defenses and immunomodulatory countermeasures by the tick. Tick feeding stimulates host immune response pathways involving antigen-presenting cells, cytokines, B-cells, T-cells, circulating and homocytotropic antibodies, granulocytes, and an array of biologically active molecules. In response to host immune defenses, tick-mediated host immunosuppressive countermeasures inhibit: host antibody responses; complement activation; T-cell proliferation; and cytokine elaboration by macrophages and Th1-lymphocytes. Immunosuppressive proteins identified in tick salivary glands and saliva have been partially characterised. Tick-induced host immunosuppression facilitates blood meal acquisition and is an important factor in the transmission/establishment of the tick-borne disease-causing agent, Borrelia burgdorferi. A novel strategy for control of tick-borne pathogens is proposed.     

Molecular Detection of Tick-Borne Pathogens in Humans with Tick Bites and Erythema Migrans,in the Netherlands

BackgroundTick-borne diseases are the most prevalent vector-borne diseases in Europe. Knowledge on the incidence and clinical presentation of other tick-borne diseases than Lyme borreliosis and tick-borne encephalitis is minimal, despite the high human exposure to these pathogens through tick bites. Using molecular detection techniques, the frequency of tick-borne infections after exposure through tick bites was estimated.MethodsTicks, blood samples and questionnaires on health status were collected from patients that visited their general practitioner with a tick bite or erythema migrans in 2007 and 2008. The presence of several tick-borne pathogens in 314 ticks and 626 blood samples of this cohort were analyzed using PCR-based methods. Using multivariate logistic regression, associations were explored between pathogens detected in blood and self-reported symptoms at enrolment and during a three-month follow-up period.ResultsHalf of the ticks removed from humans tested positive for Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Candidatus Neoehrlichia mikurensis, Rickettsia helvetica, Rickettsia monacensis, Borrelia miyamotoi and several Babesia species. Among 92 Borrelia burgdorferi s. l. positive ticks, 33% carried another pathogen from a different genus. In blood of sixteen out of 626 persons with tick bites or erythema migrans, DNA was detected from Candidatus Neoehrlichia mikurensis (n = 7), Anaplasma phagocytophilum (n = 5), Babesia divergens (n = 3), Borrelia miyamotoi (n = 1) and Borrelia burgdorferi s. l. (n = 1). None of these sixteen individuals reported any overt symptoms that would indicate a corresponding illness during the three-month follow-up period. No associations were found between the presence of pathogen DNA in blood and; self-reported symptoms, with pathogen DNA in the corresponding ticks (n = 8), reported tick attachment duration, tick engorgement, or antibiotic treatment at enrolment.ConclusionsBased on molecular detection techniques, the probability of infection with a tick-borne pathogen other than Lyme spirochetes after a tick bite is roughly 2.4%, in the Netherlands. Similarly, among patients with erythema migrans, the probability of a co-infection with another tick-borne pathogen is approximately 2.7%. How often these infections cause disease symptoms or to what extend co-infections affect the course of Lyme borreliosis needs further investigations.     

Coding nucleotide sequences of tick-borne encephalitis virus strains isolated from human blood without clinical symptoms of infection

Genomes of four tick-borne encephalitis virus strains, isolated from the blood of the individuals after tick bites and causing no clinical symptoms of infection, were characterized. Analysis of translated polypeptides revealed 21 amino acid positions typical of this group of strains and distinguishing them from the other tick-borne encephalitis virus strains of Far Eastern subtype examined earlier. Only three mutations led to substantial amino acid changes, which probably could affect the infection process severity. It is suggested that two associated mutations, deletion of amino acid 111 in the capsid protein C and substitution (Ser1534 → Phe) in the NS3 protein influence strictly coordinated polyprotein processing, disturbing correct arrangement of viral particles. This process can result in the development of defect viral particles, containing no RNA. Mutation (Ser917 → Gly) in nonstructural protein NS1 results in the substitution of hydrophilic amino acid, specific to highly virulent strains, by the hydrophobic one. This could influence the effectiveness of viral replication complex, thereby affecting the infectivity of tick-borne encephalitis virus strains.     

Correlation of TBE Incidence with Red Deer and Roe Deer Abundance in Slovenia

Tick-borne encephalitis (TBE) is a virus infection which sometimes causes human disease. The TBE virus is found in ticks and certain vertebrate tick hosts in restricted endemic localities termed TBE foci. The formation of natural foci is a combination of several factors: the vectors, a suitable and numerous enough number of hosts and in a habitat with suitable vegetation and climate. The present study investigated the influence of deer on the incidence of tick-borne encephalitis. We were able to obtain data from deer culls. Using this data, the abundance of deer was estimated and temporal and spatial analysis was performed. The abundance of deer has increased in the past decades, as well as the incidence of tick-borne encephalitis. Temporal analysis confirmed a correlation between red deer abundance and tick-borne encephalitis occurrence. Additionally, spatial analysis established, that in areas with high incidence of tick-borne encephalitis red deer density is higher, compared to areas with no or few human cases of tick-borne encephalitis. However, such correlation could not be confirmed between roe deer density and the incidence of tick-borne encephalitis. This is presumably due to roe deer density being above a certain threshold so that availability of tick reproduction hosts has no apparent effect on ticks'' host finding and consequently may not be possible to correlate with incidence of human TBE.     

蜱传脑炎病毒非结构蛋白NS2B-NS3与NS5的研究进展

蜱传脑炎病毒是引起严重的中枢神经系统疾病蜱传脑炎的病原体,每年在欧洲、俄罗斯远东地区、日本和中国北部报道的蜱传脑炎病例数约为10000-12000例,且在我国和多个欧洲国家的发病率逐渐增高,正成为人类健康的潜在危害。主动免疫是预防蜱传脑炎的有效措施,包括我国在内的多个国家已研制出安全性较高的疫苗,但在我国流行省份的疫苗接种较为有限,特异性抗病毒药物的研发或许是治疗蜱传脑炎病毒感染的研究方向之一。蜱传脑炎病毒非结构蛋白NS2B-NS3与NS5因为在病毒基因组复制、加帽和宿主免疫调节中的重要作用,成为关键的抗病毒药物研发靶点。本文综述了蜱传脑炎病毒非结构蛋白NS2B-NS3与NS5的三维结构和抑制剂研发工作,为深入探究该病毒感染的分子机制和抗病毒药物研发提供参考。     

Tick saliva in anti-tick immunity and pathogen transmission

When feeding on vertebrate host ticks (ectoparasitic arthropods and potential vectors of bacterial, rickettsial, protozoal, and viral diseases) induce both innate and specific acquired host-immune reactions as part of anti-tick defenses. In a resistant host immune defense can lead to reduced tick viability, sometimes resulting in tick death. Tick responds to the host immune attack by secreting saliva containing pharmacologically active molecules and modulating host immune response. Tick saliva-effected immunomodulation at the attachment site facilitates both tick feeding and enhances the success of transmission of pathogens from tick into the host. On the other hand, host immunization with antigens from tick saliva can induce anti-tick resistance and is seen to be able to induce immunity against pathogens transmitted by ticks. Many pharmacological properties of saliva described in ticks are shared widely among other blood-feeding arthropods.     

Altitudinal patterns of tick and host abundance: a potential role for climate change in regulating tick-borne diseases?

The impact of climate change on vector-borne infectious diseases is currently controversial. In Europe the primary arthropod vectors of zoonotic diseases are ticks, which transmit Borrelia burgdorferi sensu lato (the agent of Lyme disease), tick-borne encephalitis virus and louping ill virus between humans, livestock and wildlife. Ixodes ricinus ticks and reported tick-borne disease cases are currently increasing in the UK. Theories for this include climate change and increasing host abundance. This study aimed to test how I. ricinus tick abundance might be influenced by climate change in Scotland by using altitudinal gradients as a proxy, while also taking into account the effects of hosts, vegetation and weather effects. It was predicted that tick abundance would be higher at lower altitudes (i.e. warmer climates) and increase with host abundance. Surveys were conducted on nine hills in Scotland, all of open moorland habitat. Tick abundance was positively associated with deer abundance, but even after taking this into account, there was a strong negative association of ticks with altitude. This was probably a real climate effect, with temperature (and humidity, i.e. saturation deficit) most likely playing an important role. It could be inferred that ticks may become more abundant at higher altitudes in response to climate warming. This has potential implications for pathogen prevalence such as louping ill virus if tick numbers increase at elevations where competent transmission hosts (red grouse Lagopus lagopus scoticus and mountain hares Lepus timidus) occur in higher numbers.     

A Review of Studies on the Transmission of Anaplasma phagocytophilum from Sheep: Implications for the Force of Infection in Endemic Cycles

We review the findings of a longitudinal study of transmission of the intracellular tick-borne bacterium Anaplasma phagocytophilum from sheep to Ixodes ricinus ticks under natural conditions of tick attachment in the UK. In this study, sheep-to-tick transmission efficiency varied in a quadratic relationship with the number of adult ticks that were feeding on the sheep. We raise the hypothesis that this relationship may be due to conflicting effects of the density of ticks on bacterial survival and target cell (neutrophil) fluxes at the tick-host interface: in the same sheep at the same time, resistance to ticks was progressively inhibited with increasing number of feeding adult ticks, and investigation of serological responses to tick antigens suggesting loss of resistance may be associated with polarisation of host Th1 to Th2 type responses to ticks. We also raise the hypothesis that these properties, with superimposed effects on tick survival, may mean that variation in tick density is an important causal factor of observed variations in the force of A. phagocytophilum infection amongst different geographic foci. This revised version was published online in July 2006 with corrections to the Cover Date.     

Attachment site selection of ticks on roe deer, <Emphasis Type="Italic">Capreolus capreolus</Emphasis>

The spatio-temporal attachment site patterns of ticks feeding on their hosts can be of significance if co-feeding transmission (i.e. from tick to tick without a systemic infection of the host) of pathogens affects the persistence of a given disease. Using tick infestation data on roe deer, we analysed preferred attachment sites and niche width of Ixodes ticks (larvae, nymphs, males, females) and investigated the degree of inter- and intrastadial aggregation. The different development stages showed rather consistent attachment site patterns and relative narrow feeding site niches. Larvae were mostly found on the head and on the front legs of roe deer, nymphs reached highest densities on the head and highest adult densities were found on the neck of roe deer. The tick stages feeding (larvae, nymphs, females) on roe deer showed high degrees of intrastadial spatial aggregation, whereas males did not. Male ticks showed large feeding site overlap with female ticks. Feeding site overlap between larval-female and larval-nymphal ticks did occur especially during the months May–August on the head and front legs of roe deer and might allow pathogen transmission via co-feeding. Tick density, niche width and niche overlap on roe deer are mainly affected by seasonality, reflecting seasonal activity and abundance patterns of ticks. Since different tick development stages occur spatially and temporally clustered on roe deer, transmission experiments of tick-borne pathogens are urgently needed.