Antiplatelet-derived growth factor (PDGF) activity in the saliva of ixodid ticks is linked with their long mouthparts.

The saliva of blood-feeding arthropods modulates their vertebrate hosts' haemostatic, inflammatory and immune responses to facilitate blood feeding. In a previous study, we showed that salivary gland products from ixodid tick species also manipulate the wound-healing response by targeting at least four different mammalian growth factors: transforming growth factor ß1, hepatocyte growth factor, fibroblast growth factor 2 and platelet-derived growth factor (PDGF). In addition, species that showed PDGF-binding activity also inhibited cell proliferation in vitro and induced changes in cell morphology accompanied by disruption of the actin cytoskeleton. Here, we show a correlation between the length of the tick hypostome, the sclerotized feeding tube of the mouthparts inserted into the host's skin and anti-PDGF activity. This apparent link between hypostome length, and hence the potential depth of skin damage, and PDGF-binding activity was not apparent for the other growth factors or for other cytokines important in wound healing (keratinocyte growth factor, interleukin 6 and stromal cell-derived factor 1). However, PDGF-binding activity was no longer correlated with anti-cell activities, indicating that an additional as yet unidentified activity in tick saliva may affect cellular changes in wound repair.

Evasin-3-like anti-chemokine activity in salivary gland extracts of ixodid ticks during blood-feeding: a new target for tick control.

Ticks exploit many evasion mechanisms to circumvent the immune control of their hosts including subversion of the communication language between cells of the immune system provided by chemokines and other cytokines. One subversive molecule secreted in the saliva of Rhipicephalus sanguineus is Evasin-3, a structurally unique 7 kDa protein that selectively binds the neutrophil chemoattractants, CXCL8 and (with lower affinity) CXCL1. We compared anti-human CXCL8 and anti-mouse CXCL1/KC activities in salivary gland extracts prepared from adult Amblyomma variegatum, Rhipicephalus appendiculatus and Dermacentor reticulatus ticks during blood-feeding. Both anti-CXCL8 activity and anti-CXCL1 activity were detected in all species and in both adult females and males, with consistently higher activity levels against CXCL8. These results suggest that Evasin-3-like activity is common amongst metastriate ixodid tick species, and provide further evidence of the importance to ticks in controlling neutrophils during blood-feeding. As such, Evasin-3 offers a new target for anti-tick vaccine development.

Immunomodulatory arsenal of nymphal ticks.

Ticks have developed their own immunomodulatory mechanisms to inhibit the host inflammatory response. One of them involves the ability to subvert the cytokine network at the site of tick feeding by secreting cytokine binding molecules. Most studies have focused on the immunomodulatory prowess of adult female ticks. Here we describe anti-cytokine activity in salivary gland extracts (SGEs) prepared from 2-day-fed nymphs of Dermacentor reticulatus Fabricius, Ixodes ricinus L., Rhipicephalus appendiculatus Neumann and Amblyomma variegatum Fabricius. Anti-CXCL8 activity was detected in nymphs of all species. Relatively high activity against CCL2, CCL3 and CCL11 was observed in SGEs of R. appendiculatus and A. variegatum nymphs, whereas SGEs of I. ricinus nymphs showed comparatively high anti-interleukin-2 (-IL-2) and anti-IL-4 activities. These data show that nymphs, which epidemiologically are usually more important than adults as disease vectors, possess a range of anti-cytokine activities that may facilitate pathogen transmission.

Effects of horsefly (Tabanidae) salivary gland extracts on isolated perfused rat heart.

The speed with which horseflies (Diptera: Tabanidae) obtain a bloodmeal suggests they have potent vasodilators. We used isolated perfused rat heart to examine the vasoactivity of salivary gland extracts (SGEs) of three horsefly species, Hybomitra bimaculata Macquart, Tabanus bromius Linnaeus and Tabanus glaucopis Meigen. Administration of horsefly SGEs to the heart produced biphasic coronary responses: a decrease and subsequent increase in coronary flow (CF), characterized by initial vasoconstriction followed by prolonged vasodilation of coronary vessels. However, although SGEs of H. bimaculata induced a significant decrease in left ventricular pressure (LVP), the effect on changes in CF was not significant except at the highest dose tested. The ability to reduce LVP without significantly lowering CF, or affecting heart rate and rhythm, represents a unique set of properties that have considerable therapeutic potential if they can be reproduced by a single molecule.

Differential anti-chemokine activity of Amblyomma variegatum adult ticks during blood-feeding.

Ticks secrete a cocktail of immunomodulatory molecules in their saliva during blood-feeding, including chemokine-binding factors that help control the activity of host immunocompetent cells. Here we demonstrate differential dynamics of anti IL-8 (CXCL8), MCP-1 (CCL2), MIP-1 (CCL3), RANTES (CCL5) and eotaxin (CCL11) activities in salivary gland extracts of adult Amblyomma variegatum. Unfed male and female ticks showed activity against all the chemokines except CCL5; anti-CCL11 activity was particularly high. However, during feeding the dynamics of anti-chemokine activity differed significantly between males and females, and varied between chemokines. In males, anti-chemokine activities increased, whereas in females they declined or increased slightly as feeding progressed. The exception was anti-CCL11 activity, which declined and then increased in both males and females. Comparison of salivary gland equivalents of individual ticks prepared at various feeding intervals revealed some differences that were most pronounced between individual females fed for 8 days. These observations reflect the feeding behaviour of male and female A. variegatum. They support the concept of 'mate guarding', in which males help their mates to engorge by controlling their host's immune response, and the possibility that ticks benefit from feeding together by exploiting molecular individuality.

Impact of climate change on health: what is required of climate modellers?

The potential impacts of climate change on human health are significant, ranging from direct effects such as heat stress and flooding, to indirect influences including changes in disease transmission and malnutrition in response to increased competition for crop and water resources. Development agencies and policy makers tasked with implementing adaptive strategies recognize the need to plan for these impacts. However at present there is little guidance on how to prioritize their funding to best improve the resilience of vulnerable communities. Here we address this issue by arguing that closer collaboration between the climate modelling and health communities is required to provide the focused information necessary to best inform policy makers. The immediate requirement is to create multidisciplinary research teams bringing together skills in both climate and health modelling. This will enable considerable information exchange, and closer collaboration will highlight current uncertainties and hopefully routes to their reduction. We recognize that climate is only one aspect influencing the highly complex behaviour of health and disease issues. However we are optimistic that climate-health model simulations, including uncertainty bounds, will provide much needed estimates of the likely impacts of climate change on human health.

Exposed and concealed antigens as vaccine targets for controlling ticks and tick-borne diseases.

Tick vaccines derived from Bm86, a midgut membrane-bound protein of the cattle tick, Boophilus microplus, are currently the only commercially available ectoparasite vaccines. Despite its introduction to the market in 1994, and the recognized need for alternatives to chemical pesticides, progress in developing effective antitick vaccines (and ectoparasite vaccines in general) is slow. The primary rate-limiting step is the identification of suitable antigenic targets for vaccine development. Two sources of candidate vaccine antigens have been identified: 'exposed' antigens that are secreted in tick saliva during attachment and feeding on a host and 'concealed' antigens that are normally hidden from the host. Recently, a third group of antigens has been distinguished that combines the properties of both exposed and concealed antigens. This latter group offers the prospect of a broad-spectrum vaccine effective against both adults and immature stages of a wide variety of tick species. It also shows transmission-blocking and protective activity against a tick-borne pathogen. With the proliferation of molecular techniques and their application to vaccine development, there are high hopes for new and effective antitick vaccines that also control tick-borne diseases.

Tick-borne Great Island Virus: (II) Impact of age-related acquired immunity on transmission in a natural seabird host.

Tick-borne pathogen transmission is dependent upon tick number per host and the physical and temporal distribution of each feeding stage. Age-related acquired immunity to tick and pathogen may also be important but has received less attention. In this study we evaluate which of these parameters has the greatest impact on Great Island Virus (GIV) transmission between Ixodes uriae ticks and common guillemots (Uria aalge). The study system is well suited to investigate age-related effects because the guillemot population is naturally divided into 2 groups, older breeding and younger pre-breeding adult birds. The physical distribution and timing of adult and nymphal tick feeding was similar for both guillemot age groups. However, breeding birds were parasitized by significantly more ticks (mainly nymphs). Calculations based on tick number predict virus prevalence should be higher in ticks that have fed on breeding rather than pre-breeding birds. However, empirical evidence indicates the reverse. Protective acquired immunity to GIV infection may be the reason why GIV prevalence is actually significantly lower in ticks that have fed on breeders. Far more breeding (74%) than pre-breeding (12%) guillemots had antibodies that neutralized 1 or more GIV strains. Estimates of the force of infection support the view that pre-breeding birds experience higher rates of virus infection than breeding birds. The results indicate age-related acquired immunity is a key factor in GIV transmission and highlight the need to consider age-related effects and host immunity when undertaking quantitative studies of tick-borne pathogen transmission.

Tick-borne Great Island Virus: (I) Identification of seabird host and evidence for co-feeding and viraemic transmission.

Great Island Virus (GIV) is an arbovirus present in the tick Ixodes uriae, a common ectoparasite of nesting seabirds. Common guillemot (Uria aalge) and black-legged kittiwake (Rissa tridactyla) are the preferred and most abundant hosts of I. uriae on the Isle of May, Scotland. As part of a study to understand the epidemiology of GIV, the ability of guillemot and kittiwake to support tick-borne transmission of GIV was examined. GIV was present in ticks feeding in isolated guillemot colonies and guillemots had virus-specific neutralizing antibodies demonstrating previous GIV infection. By contrast, only uninfected ticks were found in colonies inhabited solely by kittiwakes. GIV was isolated from kittiwake ticks in colonies which also contained breeding guillemots but no virus-specific neutralizing antibodies were present in blood samples of kittiwake on which infected ticks were feeding. Thus guillemots are the main vertebrate hosts of GIV on the Isle of May whereas kittiwakes do not appear to be susceptible to infection. Virus infection of adult ticks feeding on guillemots was highly efficient and may involve both viraemic transmission and transmission from infected to uninfected ticks feeding together on birds that do not develop a patent viraemia.

Manipulation of host cytokine network by ticks: a potential gateway for pathogen transmission.

Ticks are obligatory blood-feeding arthropods that secrete various immunomodulatory molecules to antagonize host inflammatory and immune responses. Cytokines play an important role in regulating these responses. We investigated the extent to which ticks interact with the sophisticated cytokine network by comparing the effect of salivary gland extracts (SGE) of 3 ixodid tick species, Dermacentor reticulatus, Amblyomma variegatum and Ixodes ricinus, all of which are important vectors of tick-borne pathogens. Using specific ELISAs, anti-cytokine activity was demonstrated with 7 cytokines: IL-8, MCP-1, MIP-1alpha, RANTES, eotaxin, IL-2 and IL-4. The results varied between species, and between adult males and females of the same species. Relatively high activity levels were detected in saliva of female D. reticulatus, confirming that the observed anti-cytokine activities are an integral part of tick saliva secreted into the host. Results with fractionated SGE indicated that from 2 to 6 putative cytokine binding molecules are produced, depending on species and sex. Binding ability of SGE molecules was verified by cross-linking with radio-isotope labelled MIP-1alpha. By targeting different cytokines, ixodid ticks can manipulate the cytokine network, which will greatly facilitate blood-feeding and provide a gateway for tick-borne pathogens that helps explain why ticks are such efficient and effective disease vectors.

Tick-host interactions: saliva-activated transmission.

The skin site at which ticks attach to their hosts to feed is the critical interface between the tick and its host, and tick-borne pathogens. This site is highly modified by the pharmacologically active molecules secreted in tick saliva. For pathogens, it is an ecologically privileged niche that many exploit. Such exploitation is referred to as saliva-activated transmission (SAT) - the indirect promotion of tick-borne pathogen transmission via the actions of bioactive tick saliva molecules on the vertebrate host. Here we review evidence for SAT and consider what are the most likely candidates for SAT factors among the tick pharmacopoeia of anti-haemostatic, anti-inflammatory and immunomodulatory molecules identified to date. SAT factors appear to differ for different pathogens and tick vector species, and possibly even depend on the vertebrate host species. Most likely we are searching for a suite of molecules that act together to overcome the redundancy in host response mechanisms. Whatever they turn out to be, the quest to identify the tick molecules that mediate SAT is an exciting one, and offers new insights to controlling ticks and tick-borne diseases.

Tick-borne viruses.

At least 38 viral species are transmitted by ticks. Virus-tick-vertebrate host relationships are highly specific and less than 10% of all tick species (Argasidae and Ixodidae) are known to play a role as vectors of arboviruses. However, a few tick species transmit several (e.g. Ixodes ricinus, Amblyomma variegatum) or many (I. uriae) tick-borne viruses. Tick-borne viruses are found in six different virus families (Asfarviridae, Reoviridae, Rhabdoviridae, Orthomyxoviridae, Bunyaviridae, Flaviviridae) and at least 9 genera. Some as yet unassigned tick-borne viruses may belong to a seventh family, the Arenaviridae. With only one exception (African swine fever virus, family Asfarviridae) all tick-borne viruses (as well as all other arboviruses) are RNA viruses. Tick-borne viruses are found in all the RNA virus families in which insect-borne members are found, with the exception of the family Togaviridae. Some tick-borne viruses pose a significant threat to the health of humans (Tick-borne encephalitis virus, Crimean-Congo haemorrhagic fever virus) or livestock (African swine fever virus, Nairobi sheep disease virus). Key challenges are to determine the molecular adaptations that allow tick-borne viruses to infect and replicate in both tick and vertebrate cells, and to identify the principal ecological determinants of tick-borne virus survival.

Dynamics of infection in tick vectors and at the tick-host interface.

Tick-borne flaviviruses are common, widespread, and successfully adapted to their mode of transmission. Most tick vectors of flaviviruses are ixodid species. These ticks are characterized by a comparatively long life cycle, lasting several years, during which the infecting virus may be maintained from one developmental stage of the tick to the next. Hence ticks act as highly efficient reservoirs of flaviviruses. Many tick-borne flaviviruses are transmitted vertically, from adult to offspring, although the frequency is too low to maintain the viruses solely in the tick population. Instead, the survival of tick-borne flaviviruses is dependent on horizontal transmission, both from an infected tick to a susceptible vertebrate host and from an infected vertebrate to uninfected ticks feeding on the animal. The dynamics of transmission and infection have traditionally been considered in isolation: in the tick, following virus uptake in the infected blood meal, infection of the midgut, passage through the hemocoel to the salivary glands, and transmission via the saliva; and in the vertebrate host, virus delivery into the skin at the site of tick feeding, infection of the draining lymph nodes, and dissemination to target organs. However, there is now compelling evidence of a complex interaction between the tick vector and its vertebrate host that affects virus transmission profoundly. The feeding site in the skin is a battleground in which the hemostatic, inflammatory, and immune responses of the host are countered by antihemostatic, anti-inflammatory, and immunomodulatory molecules (mostly proteins and peptides) secreted in tick saliva. Here we speculate that exploitation of the tick pharmacopeia, rather than development of viremia, is the key step in successful tick-borne flavivirus transmission.

Vasodilatory activity in horsefly and deerfly salivary glands.

Salivary gland extract (SGE) of four horsefly species (Hybomitra bimaculata Macquart, Hybomitra ciureai Séguy, Tabanus bromius L., Tabanus glaucopis Meigen) and one deerfly species (Chrysops relictus Meigen) (Diptera: Tabanidae) were shown to contain vasodilatory activity. Aliquots equivalent to 1, 5 and 10 pairs of salivary glands (SG) relaxed rat femoral artery (with intact endothelium) pre-constricted with phenylephrine. Vasodilatory activity was dose-dependent. SGE of one horsefly species (Haematopota pluvialis L.) did not induce relaxation. The kinetics of vasodilation induced by SGE of four horsefly species differed from the deerfly. These results indicate that tabanid species may produce more than one type of vasodilator to aid blood feeding.

Tick-borne flaviviruses.

Tick-borne encephalitis (TBE), one of the most dangerous neuroinfections in Europe and Asia, is caused by tick-borne encephalitis virus (TBEV) and currently involves approximately 11,000 human cases annually, mostly in Russia. This chapter describes the main problems associated with the epidemiology, ecology, pathogenesis, and control of this disease. We have attempted to review the factors that influence the incidence and distribution of TBE, and to discuss possible reasons for the different clinical manifestations including most commonly observed asymptomatic infections, fever forms, acute encephalitis, and the less frequently registered biphasic milk fever and chronic encephalitis. Epidemiologic data concerning the other tick-borne flaviviruses, namely Louping ill virus, Langat virus, and Powassan virus that also produce encephalitis on a smaller scale, are also presented. Here we describe the history and current epidemiological role of Omsk hemorrhagic fever virus and Kyasanur forest disease virus, two viruses that are genetically closely related to TBEV, but produce hemorrhagic fever instead of encephalitis, and provide possible explanations for these differences. The other viruses in the tick-borne flavivirus group are also included despite the fact that they do not play an essential epidemiologic role in humans. This chapter contains a brief history of vaccination against TBE including the trials with live attenuated vaccine and reviews the modern trends in development of vaccine virus strains.

Vesicular stomatitis virus nucleocapsid protein production in cells treated with selected fast protein liquid chromatography fractions of tick salivary gland extracts.

A salivary gland extract (SGE) prepared from 5-days-fed Dermacentor reticulatus female ticks was fractionated by fast protein liquid chromatography (FPLC). The effect of three FPLC fractions selected on the basis of anti-interleukin 8 (anti-IL-8) activity on vesicular stomatitis virus (VSV) nucleocapsid (N) protein formation in mouse L-cells was determined. Infected 14C-labeled cells treated with the FPLC fractions were analyzed by two-dimensional (2D) electrophoresis. The yields of VSV N protein were evaluated by Imagemaster software analysis. Most noticeable was an increase in the N protein production after treatment with the fraction 39 corresponding to the major peak of the anti-IL-8 activity. The nature of the substance in SGE that was responsible for this effect remains unclear.

Anticoagulant activities in salivary glands of tabanid flies.

Tabanid flies are telmophages (pool feeders), taking frequent and rapid bloodmeals from many different individual hosts. To investigate how they accomplish this intermittent feeding strategy, we examined the anticoagulant activities in salivary gland extracts (SGE) from 19 species representing six genera: Atylotus, Chrysops, Haematopota, Heptatoma, Hybomitra and Tabanus (Diptera: Tabanidae). Standard coagulation screen assays were used to determine thrombin time, prothrombin time and activated partial thromboplastin time. Chromogenic substrate assays were performed for thrombin and factor Xa activity. SGE of most species (except Chrysops spp.) considerably prolonged human plasma clotting time in a dose-dependent manner, and showed potent and specific antithrombin activity in the chromogenic substrate assay. Heptatoma pellucens displayed the strongest anticoagulant activity. Specific anti-factor Xa activity in tabanid SGE was not detected. Electrophoretic profiles of SGE proteins differed between genera and species. Overall, the results suggest that tabanids have evolved at least two antihaemostatic strategies.

Heterogeneity in the effect of different ixodid tick species on human natural killer cell activity.

Tick saliva plays a vital role in blood-feeding, including manipulation of the host response to tick infestation. Furthermore, a diverse number of tick-borne pathogens are transmitted to vertebrate hosts via tick saliva, some of which exploit the immunomodulatory activities of their vector's saliva. We report that salivary gland extracts (SGE) derived from Dermacentor reticulatus adult ticks induce a decrease in the natural killer (NK) activity of effector cells obtained from healthy human blood donors. The decrease was observed with SGE from both female and male D. reticulatus fed for either 3 or 5 days on mice, but no significant effect was observed with SGE from unfed ticks or ticks that had fed for 1 day. These results indicate that the tick anti-NK factor(s) is only active after blood-feeding has commenced. Microscopic examination revealed that the first step of NK activity, namely effector/target cell conjugate formation, was affected by SGE. The observed reduction in conjugate formation occurred when effector (but not target) cells were treated with SGE for 30 min, and the effect persisted after 12 h of treatment. Similar but less potent anti-NK activity was detected for SGE from Amblyomma variegatum and Haemaphysalis inermis. By contrast, SGE derived from Ixodes ricinus and Rhipicephalus appendiculatus female ticks did not decrease NK activity. The apparent absence of such activity in these two important vectors of tick-borne viruses suggests that control of NK cells does not play an important role in promoting virus transmission, at least for these particular species.

A high affinity serotonin- and histamine-binding lipocalin from tick saliva.

To overcome the inflammatory response in its host, the cattle-feeding, brown ear tick secretes histamine-binding proteins into the feeding site. These proteins are beta-barrels with two internal binding sites: a high-affinity (H) site for histamine and a site (L) for which the natural ligand is unknown. Here we report a related protein (SHBP), secreted by a rodent- and cattle-feeding tick, that traps both histamine and serotonin. The histamine-binding H site is well conserved in SHBP, whereas residue changes in the L-like site are consistent with binding of the bulkier serotonin molecule. As histamine is a key inflammatory mediator in cattle, while serotonin takes on this role in rodents, the diversification of these tick proteins may reflect host adaptation.

Antigenic profile of Ixodes ricinus: effect of developmental stage, feeding time and the response of different host species.

Antigens recognized by host species in response to ectoparasite infestation have been widely reported. Although differences in the immune responses of different host species have been described, only a very few of these studies compare the range of antigens recognized by different host species in response to infestation. We used Western blot analysis to investigate antigenic responses of different host species that were repeatedly infested with Ixodes ricinus ticks. Antigenic profiles of larval and nymphal whole tick homogenates were compared with the respective salivary gland extract (SGE) samples using sera from rabbits repeatedly infested with either adults, nymphs or larvae. SGE samples were also analysed using sera from hamsters infested with adults, nymphs or larvae. Sera from BALB/C mice, Apodemus flavicollis (yellow-necked mouse) or Clethrionomys glareolus (bank vole) repeatedly infested with larvae were used to compare the antigenic profiles of SGE and larval homogenate samples. We also investigated different sources of tick antigens, using rabbit sera, by comparing midgut extracts from female adult ticks and SGE from unfed ticks and from ticks throughout the 6-day feeding period with whole tick homogenates of female and male adults, nymphs and larvae. The pattern of antigenic tick-molecules recognized by infested host species varies with the period of feeding, developmental stage and the particular host species parasitized.