Disaggregation of aggregated platelets by savignygrin,a alphaIIbeta3 antagonist from Ornithodoros savignyi

Ticks control their host's hemostatic system by secretion of bioactive components during feeding that inhibit blood coagulation and platelet aggregation. Dissolution of platelets that have already aggregated can enhance control over the hemostatic system. It has been shown that disaggregation of aggregated platelets by the enzyme apyrase was accompanied by a shape change from the aggregated spherical form back to the discoid form associated with un-activated platelets. The present study concerns the disaggregation effect of the α_IIbβ₃ antagonist, savignygrin. Aggregated platelets that were disaggregated by savignygrin and platelets pre-incubated with savignygrin before activation with ADP, retained a spherical form similar to platelets disaggregated by the fibrinogenolytic enzyme plasmin. The number of pseudopods were however, markedly reduced suggesting a disruption of the focal adhesion points that act as a localization point of α_IIbβ₃. These results are concurrent with targeting of α_IIbβ₃ and dissociation of fibrinogen from its receptor, once aggregation has taken place. This is the second mediator of platelet disaggregation found in soft ticks and suggests that disaggregation of aggregated platelets might play an important part in the anti-hemostatic strategy of ticks. This revised version was published online in July 2006 with corrections to the Cover Date.     

Apyrase Activity and Platelet Aggregation Inhibitors in the Tick Ornithodoros Savignyi (Acari: Argasidae)

Ticks are ectoparasites that cause considerable damage to their hosts while feeding. The feeding process is facilitated by anti-haemostatic factors present in the tick saliva. Apyrase (ATP diphosphohydrolase, EC 3.6.1.5) is a platelet aggregation inhibitor found in most haematophagous organisms studied. The present study describes the identification and characterization of such an activity in the tick Ornithodoros savignyi. The enzyme conformed to many properties common to apyrases. These included a low substrate specificity, dependence on bivalent metal ions for activity and insensitivity to the classical ATPase inhibitors. Heat denaturation studies, pH optima and similar effects of inhibitors on the enzyme's ATP and ADP hydrolysing activities supported its classification as an apyrase. Salivary gland extracts inhibited the platelet aggregation induced by ADP, collagen and thrombin and disaggregated aggregated platelets. The results suggest the presence of two or more anti-platelet factors present in the salivary glands of this tick species.     

Plasmin-mediated macrophage reversal of low density lipoprotein aggregation

Evidence suggests that aggregated low density lipoprotein (AgLDL) accumulates in atherosclerotic lesions. Previously, we showed that AgLDL induces and enters surface-connected compartments (SCC) in human monocyte-derived macrophages by a process we have named patocytosis. Most AgLDL taken up by these macrophages in the absence of serum is stored in SCC and remains undegraded. We now show that macrophages released AgLDL (prepared by vortexing or treatment with phospholipase C or sphingomyelinase) from their SCC when exposed to 10% human lipoprotein-deficient serum (LPDS). Macrophages also took up AgLDL in the presence of LPDS, but subsequently released it. In both cases, the released AgLDL was disaggregated. Although the AgLDL that macrophages took up could not pass through a 0.45-micrometer filter, >60% of AgLDL could pass this filter after release from the macrophages. Disaggregation of AgLDL was verified by gel-filtration chromatography and electron microscopy that also showed particles larger than LDL, reflecting fusion of LDL that aggregates. The factor in serum that mediated AgLDL release and disaggregation was plasmin generated from plasminogen by macrophage urokinase plasminogen activator. AgLDL release was decreased >90% by inhibitors of plasmin (epsilon-amino caproic acid and anti-plasminogen mAb), and also by inhibitors of urokinase plasminogen activator (plasminogen activator inhibitor-1 and anti-urokinase plasminogen activator mAb). Moreover, plasminogen could substitute for LPDS and produce similar macrophage release and disaggregation of AgLDL. Because only plasmin bound to the macrophage surface is protected from serum plasmin inhibitors, interaction of AgLDL with macrophages was necessary for reversal of its aggregation by LPDS. The released disaggregated LDL particles were competent to stimulate LDL receptor-mediated endocytosis in cultured fibroblasts. Macrophage-mediated disaggregation of aggregated and fused LDL is a mechanism for transforming LDL into lipoprotein structures size-consistent with lipid particles found in atherosclerotic lesions.     

Saliva of the soft tick, Ornithodoros moubata, contains anti-platelet and apyrase activities.

1. Pilocarpine-induced saliva of the soft tick Ornithodoros moubata inhibits platelet aggregation induced by ADP or collagen, even when diluted 2000 times into platelet rich plasma. 2. Saliva contains apyrase (ATP-diphosphohydrolase) activity, which has an optimal pH of 7.0 for ADP and of 8.0 for ATP hydrolysis, respectively. Both Ca2+ and Mg2+ activate the reactions. 3. The mean specific activities for ATP and ADP hydrolysis at pH 7.5 were 0.97 and 0.74 mumoles orthophosphate/min/mg protein. 4. These results, which demonstrate for the first time such activities in the saliva of soft ticks, support the hypothesis that the saliva of blood sucking arthropods serves an anti-hemostatic role during feeding and that large amounts of salivary apyrase activity have evolved independently in hematophagous arthropods.     

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.     

The seabird tick, <Emphasis Type="Italic">Ixodes</Emphasis><Emphasis Type="Italic">uriae</Emphasis>, uses uric acid in penguin guano as a kairomone and guanine in tick feces as an assembly pheromone on the Antarctic Peninsula

In the vicinity of Palmer Station, Antarctica, the seabird tick, Ixodes uriae, forms large aggregations under rocks at the periphery of Adelie penguin rookeries. When the adult penguins return to the rookeries at the beginning of the nesting season the ticks leave their off-host aggregation site, attach to the penguins for a period of feeding, and then subsequently return to the aggregation site. In this study, we searched for chemical cues that may be used by the ticks to locate their aggregation sites as well as cues involved in finding penguins. Tick excreta and soil extracts from beneath tick aggregations contained a pheromone that elicited assembly behavior in unfed larvae, non-fed nymphs and non-fed adults. Guanine, the major excretory product of ticks, elicited assembly behavior, thus, guanine is likely an active component involved in assembly. Non-fed stages also responded positively to penguin guano and uric acid, the primary excretory product of penguins, suggesting that uric acid and other components of penguin guano function as a kairomone used by the non-fed ticks to locate their host. After feeding, the immature ticks’ response to both the assembly and kairomones is switched off for several days, and the ticks regain responsiveness only after they have molted. Fed adult females lay eggs and die without ever regaining responsiveness. Thus, I. uriae relies on two closely related chemicals to regulate two critical aspects of its life: assembly and host-finding. Guanine and other components of tick excreta function as an assembly pheromone in promoting the formation of off-host aggregations, while uric acid and other components of penguin guano function as a kairomone used by the tick to locate its host.     

Saliva-activated transmission (SAT) of Thogoto virus: dynamics of SAT factor activity in the salivary glands ofRhipicephalus appendiculatus,Amblyomma variegatum,andBoophilus microplus ticks

Thogoto (THO) virus is transmitted from infected to uninfected ticks when co-feeding on uninfected guinea-pigs, even though the guinea-pigs do not develop a detectable viraemia. This form of non-viraemic transmission is potentiated by a factor (s) secreted by the saliva of ticks and hence has been termed saliva-activated transmission (SAT). The synthesis of the SAT factor by the salivary glands of three ixodid tick species was determined by placing uninfected nymphal ticks on guineapigs that were subsequently inoculated with a mixture of THO virus and salivary gland extract (SGE) derived from one of the tick species. SAT factor activity was measured by determining the number of nymphs that acquired THO virus. For the three-host ixodid species,Rhipicephalus appendiculatus andAmblyomma variegatum, maximum enhancement of THO virus transmission was observed when salivary glands were derived from uninfected, female ticks that had fed for a period of 6 or 8 days, respectively. In contrast, when salivary glands were derived form uninfected femaleBoophilus microplus, a one-host ixodid tick species, enhancement of THO virus transmission was observed throughout the tick feeding period. Thus, the natural feeding behaviour of ticks appears to be an important factor in determining the relative importance of these vectors in mediating SAT.     

N-Ethylmaleimide induces disaggregation of platelets preaggregated by ADP and thrombin and decreases cytoplasmic calcium level

The effect of N-ethylmaleimide (NEM, 1-200 microM) on ADP- and thrombin-induced platelet aggregation and thrombin-induced increase in intracellular Ca2+ concentration was studied. Addition of NEM to platelets preaggregated with ADP or thrombin induces platelet disaggregation. The anti-aggregant activity of NEM was different for ADP- and thrombin-induced aggregations. At 200 microM concentration, NEM completely disaggregated ADP-induced aggregates and only partially disaggregated thrombin-aggregated platelets. NEM did not influence the thrombin-induced increase in cytoplasmic Ca2+ and had no effect on the basal level of Ca2+ in the cytosol of non-activated platelets. However, NEM decreased the level of thrombin-mobilized Ca2+ in the cytosol of activated platelets. Thus, NEM can induce disaggregation of ADP- and thrombin-preaggregated platelets by activating a system which removes Ca2+ from the platelet cytosol.     

A blood meal-induced Ixodes scapularis tick saliva serpin inhibits trypsin and thrombin,and interferes with platelet aggregation and blood clotting

Ixodes scapularis is a medically important tick species that transmits causative agents of important human tick-borne diseases including borreliosis, anaplasmosis and babesiosis. An understanding of how this tick feeds is needed prior to the development of novel methods to protect the human population against tick-borne disease infections. This study characterizes a blood meal-induced I. scapularis (Ixsc) tick saliva serine protease inhibitor (serpin (S)), in-house referred to as IxscS-1E1. The hypothesis that ticks use serpins to evade the host’s defense response to tick feeding is based on the assumption that tick serpins inhibit functions of protease mediators of the host’s anti-tick defense response. Thus, it is significant that consistent with hallmark characteristics of inhibitory serpins, Pichia pastoris-expressed recombinant IxscS-1E1 (rIxscS-1E1) can trap thrombin and trypsin in SDS- and heat-stable complexes, and reduce the activity of the two proteases in a dose-responsive manner. Additionally, rIxscS-1E1 also inhibited, but did not apparently form detectable complexes with, cathepsin G and factor Xa. Our data also show that rIxscS-1E1 may not inhibit chymotrypsin, kallikrein, chymase, plasmin, elastase and papain even at a much higher rIxscS-1E1 concentration. Native IxscS-1E1 potentially plays a role(s) in facilitating I. scapularis tick evasion of the host’s hemostatic defense as revealed by the ability of rIxscS-1E1 to inhibit adenosine diphosphate- and thrombin-activated platelet aggregation, and delay activated partial prothrombin time and thrombin time plasma clotting in a dose-responsive manner. We conclude that native IxscS-1E1 is part of the tick saliva protein complex that mediates its anti-hemostatic, and potentially inflammatory, functions by inhibiting the actions of thrombin, trypsin and other yet unknown trypsin-like proteases at the tick–host interface.     

Ixodes dammini: evidence for salivary prostacyclin secretion

Pilocarpine-induced saliva of adult Ixodes dammini ticks contained abundant amounts (523 +/- 140 ng/ml, mean +/- SE, n = 14) of 6-keto-PGF1 alpha, the stable degradation product of prostacyclin. This prostaglandin was identified by radioimmunoassay and reversed-phase chromatography. This activity may help tick feeding by preventing host hemostatic reactions, by increasing host blood flow at the tick feeding site, and by preventing leukocyte degranulation.     

Chemical attraction of Dermacentor variabilis ticks parasitic to Peromyscus leucopus based on host body mass and sex

Macroparasites are commonly aggregated on a small subset of a host population. Previous explanations for this aggregation relate to differences in immunocompetence or the degree to which hosts encounter parasites. We propose active tick host choice through chemical attraction as a potential mechanism leading to aggregated tick burdens. We test this hypothesis using a Y-maze olfactometer, comparing chemical attraction responses of larval and nymphal Dermacentor variabilis ticks parasitic to the white-footed mouse, Peromyscus leucopus, as a function of host sex and host body mass. We hypothesized that larger hosts and male hosts would be most attractive to searching ticks, as these hosts commonly have higher tick burdens in the field. Chemical attraction trials were run in the presence and absence of a known tick attractant, host-produced carbon dioxide (CO₂). Male hosts and larger hosts were preferred by nymphal D. variabilis in the presence and absence of CO₂, whereas larvae had no detectable host preference. The current study suggests that host-produced chemical cues may promote aggregated tick burdens among hosts of a single species based on host body mass and sex.     

The effect of spatial heterogenity on the aggregation of ticks on white-footed mice

Parasites are often aggregated on a minority of the individuals in their host populations. Although host characteristics are commonly presumed to explain parasite aggregation on hosts, spatio-temporal aggregation of parasites during their host-seeking stages may have a dominant effect on the aggregation on hosts. We aimed to quantify, using mixed models, repeatability and autocorrelation analyses, the degree to which the aggregation of blacklegged ticks (Ixodes scapularis) on white-footed mice (Peromyscus leucopus) is influenced by spatio-temporal distributions of the host-seeking ticks and by heterogeneity among mice. Host-seeking ticks were spatially aggregated at both the larval and nymphal life-stages. However, this spatial aggregation accounted for little of the variation in larval and nymphal burdens observed on mice (3% and 0%, respectively). Conversely, mouse identity accounted for a substantial proportion of the variance in tick burdens. Mouse identity was a significant explanatory factor as the majority of ticks parasitized a consistent set of mice throughout the activity seasons. Of the characteristics associated with mouse identity investigated, only gender affected larval burdens, and body mass and home range sizes in males were correlated with nymphal burdens. These analyses suggest that aggregation of ticks on a minority of mice does not result from the distribution of host-seeking ticks but from characteristics of the hosts.     

Brain factor induced formation of inositol phosphates in tick salivary glands

A factor from tick brain increases inositol phosphates in isolated, whole tick salivary glands. The factor is sensitive to trypsin and heat (5 min, 100°C) suggesting that it may be a neuropeptide or protein. The salivary glands undergo growth and differentiation accompanied by considerable proliferation of plasma membranes during tick feeding. Salivary glands from ticks in later stages of feeding produce higher levels of inositol phosphates than glands from ticks in early stages of feeding. Cyclic AMP modulates the formation of inositol phosphates suggesting interaction of salivary gland function by the transducing system that employs cyclic AMP as a “second messenger” and that which employs inositol phosphates.     

Interstadial variation in the attachment sites of Ixodes ricinus ticks on sheep

The spatial aggregation of ticks feeding on vertebrate hosts has been recognized for some time but, for hosts supporting more than one stage of the tick, observations of interstadial variation in the site of attachment have not previously been quantified. This study showed that all three parasitic stages of Ixodes ricinus ticks feeding on sheep attach most commonly to the hair-covered areas of the head and limbs while few ticks attach to the fleeced region of the body. However, significant differences were observed in the site of attachment of the three feeding stages of the tick. Larvae attached to distal limbs and rostral areas of the head and adult females attached to the proximal areas of the limbs and around the neck and ears, while nymphs attached in locations between the larvae and adults. The importance of the spatial aggregation of the ticks and interstadial variation in their distribution on the host, for the transmission of tick-borne pathogens and the epidemiology of the diseases they cause, is discussed. © Rapid Science Ltd. 1998     

Feeding preferences of the red‐billed oxpecker,Buphagus erythrorhynchus: a parasitic mutualist?

The oxpecker–ungulate association of sub‐Saharan Africa is an example of a complicated interspecific association subject to variation in outcome. Oxpeckers (Buphagus spp.) are unusual birds because they not only glean ticks from an array of African ungulates, but they are one of the few avian species known to wound‐feed from their living hosts. The conditions under which oxpeckers wound‐feed and the mechanisms generating variation in this association are unclear. We took a unique approach to studying the relationship by conducting a series of feeding preference experiments on twelve captive red‐billed oxpeckers (B. erythrorhynchus). We assessed how oxpecker feeding behaviour is influenced by changes in tick abundance and tick type. In cafeteria‐style experiments, oxpeckers fed equally on ticks and liquid bovine blood. In experiments using donkeys as the host animal, oxpeckers spent more time wound‐feeding when a less‐preferred tick type was available and when tick abundance was low compared to when a preferred tick type was available and when tick abundance was high. However, oxpeckers still wound‐fed even when offered a large number of the ticks they prefer. Additional experiments incorporating tick species of different stages and multiple ungulate species are necessary to fully reveal the dynamics of this association.     

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.     

Fibrinogen distribution on surfaces and in organelles of ADP stimulated human blood platelets

The fibrinogen distribution in platelet organelles after ADP-stimulation was investigated with anti-human fibrinogen using protein A-gold applied to serial sections. Fibrinogen was detected in the so-called alpha-granules of platelets and also in granule protrusions which were observed after ADP-stimulation. The ends of these protrusions were formed as coated membranes and the tips were often in apposition to the surface connected membranes or the plasmalemma. At such places fusion events and hence signs of an exocytosis could be demonstrated by means of cryofixation and cryosubstitution. Examination of serial sections revealed fibrinogen on all these granule profiles. Surface connected membranes, free surfaces and the characteristic structure of the contact zones of aggregated platelets were also labelled by gold particles but less than anticipated. On the platelet surfaces and surface connected membranes fibrinogen was rarely demonstrable with ferritin-labelled anti-human fibrinogen on washed or thrombin-stimulated, almost fibrinogen free platelets. After addition of human fibrinogen to the thrombin stimulated and disaggregated platelets a part of the platelets aggregated spontaneously and formed characteristic contact zones. Anti-human fibrinogen was observed on the free surfaces, in filamentous bridges between the contact spaces and in a tubular surface connected membrane system with involvement of coated membranes at the central ends of these structures. The results indicate the following: all alpha-granules contain fibrinogen; after ADP-stimulation secretion takes place with involvement of coated membranes; during aggregation fibrinogen binds to platelet surfaces and forms contact spaces; fibrinogen is taken up by the surface connected system with involvement of coated membranes.     

Investigations into lymphocyte transformation and histamine release by basophils in sheep repeatedly infested with Rhipicephalus evertsi evertsi ticks

The immune response of a natural host of Rhipicephalus evertsi evertsi to feeding by this tick species was investigated with respect to the effects of tick salivary gland extracts on the transformation of peripheral blood lymphocytes and the release of histamine by basophils obtained from repeatedly infested sheep.The results indicated that there was no stimulation of lymphocyte transformation but that histamine release was elevated 10 fold after four infestations.Although this suggests a hypersensitivity reaction, believed to be a major factor in resistance to tick feeding, it was observed that ticks fed normally even after four infestations with 28 day intervals in between. These results emphasize the adaptation of ticks to feeding on their natural hosts.     

Characterization of anti-hemostatic factors in the argasid, Argas monolakensis: implications for the evolution of blood-feeding in the soft tick family

To date, the only anti-hemostatic factors characterized for softs ticks are for Ornithodoros moubata and Ornithodoros savignyi, ticks that feeds mainly on mammals. This includes thrombin (ornithodorin and savignin), fXa (TAP and fXaI) and platelet aggregation (disagegin and savignygrin) inhibitors that belong to the BPTI-Kunitz protein family. This raises the question on how well anti-hemostatic factors will be conserved in other soft tick genera that feeds on other vertebrates such as birds. We characterized the anti-hemostatic factors from Argas monolakensis, a soft tick that feeds mainly on Californian gulls. The main anti-clotting factor (monobin) is an ortholog of ornithodorin and savignin and shows similar slow tight-binding kinetics. The main anti-platelet activities are apyrase and fibrinogen receptor antagonists (monogrins). The monogrins are orthologs of disagregin and savignygrin and like savignygrin presents the RGD integrin-recognition motif on the BPTI substrate-binding presenting loop. This implies that the anti-hemostatic factors evolved in the ancestral soft tick lineage and has been maintained in soft tick species from two distinct genera with different host preferences. The Argas derived anti-hemostatic factors bind to mammalian targets with affinities similar to that observed for their orthologs in the Ornithodoros genus. This cross-reactivity could have facilitated the switching of soft ticks from avian to mammalian hosts and can explain in part the ability of Argas ticks, to feed on humans, thereby remaining a possible health risk.