Characterization of Ixophilin,A Thrombin Inhibitor from the Gut of Ixodes scapularis

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.     

Cutaneous and systemic cellular responses induced by the feeding of the argasid tick Ornithodoros parkeri

Carolyn M. Johnston and Stephen J. Brown 1985. Cutaneous and systemic cellular responses induced by the feeding of the argasid tick Ornithodoros parkeri. International Journal for Parasitology15: 621–628. Initial feeding by Ornithodoros parkeri ticks induced a significant blood basophilia in guinea pigs, with a minimal cutaneous basophil response. Hosts challenged 14 days later, however, exhibited significantly depressed blood basophil levels associated with a marked accumulation of these cells at tick feeding sites in the tissue. Blood eosinophil levels in primary and secondary hosts were comparable, but eosinophil levels at tick feeding sites in challenged animals were significantly greater than levels in primary hosts. Furthermore, challenge tick feeding resulted in the activation of primary tick feeding sites on the opposite flank that became erythematous 90 min after challenge and indurated within 24 h. Histologically, these activated primary feeding sites 90 min after challenge on the opposite flank were marked by a dominant eosinophil response (314 ± 128 cells, 59% of the infiltrate) with a marked basophil component (145 ± 67 cells, 28% of the infiltrate) that resembled the active challenge feeding sites 24 h after infestation (24 ± 52 cells, 76% of the infiltrate); 90 min after challenge active feeding sites had a weak basophil response (2 ± 1 cells) similar to uninfested controls. These results suggest the chronic nature of tick bites with an apparent continual recruitment of basophils that is probably a result of slow antigen release over time by appropriately sensitized antigen presenting cells. Primary tick feeding sites in guinea pigs previously exposed to Xenopsylla cheopis fleas, on the opposite flank, contained a marked eosinophilia (63 ± 25 cells) compared to primary tick feeding sites in naive guinea pigs (2 ± 0 cells); suggesting the possibility of cross reactivity between flea and tick antigens     

Disrupting the Amblyomma americanum (L.) CD147 receptor homolog prevents ticks from feeding to repletion and blocks spontaneous detachment of ticks from their host

The CD147 receptor is a cell-surface glycoprotein in the IgG family that plays pivotal roles in intercellular interactions involved with numerous physiological and pathological processes such as extracellular matrix remodeling. We previously found an Amblyomma americanum (Aam) tick CD147 receptor homolog among genes that were up regulated in response to tick feeding stimuli. This study characterizes an AamCD147 receptor protein that is 72–83% conserved in other tick species and possess characteristic CD147 receptor sequence features: an extracellular (EC) region containing two IgG domains, a transmembrane and the cytoplasmic domains. Likewise, the AamCD147 EC domain folds into secondary structures that are consistent to the human homolog: an amino-terminus β-barrel that is linked to 2-carboxy-terminus β-sheets with consensus disulfide bonds conserved in each of the 2 domains. CD147 receptor signaling and regulatory mechanisms are putatively conserved in ticks as revealed by in silico analysis that show presence in the tick genome of CD147 receptor signaling protein homologs, cyclophilin (CyP) A and B, and chaperones that transport it to the plasma membrane, caveolin-1 and CyP60. The AamCD147 receptor has a dichotomous expression pattern of where it is up regulated in response to feeding in the salivary gland but remains constant at the midgut and ovary levels suggesting that it may regulate different functions in different tick organs. We speculate that biological functions of the AamCD147 receptor are essential to tick feeding success as revealed by RNAi-mediated silencing that caused ticks to obtain smaller blood meals, of which ～69% were below threshold to trigger spontaneous detachment of ticks from the host. These ticks showed unusual cuticle tenderness and assumed a reddish coloration, a phenomenon that has been attributed to tick midgut damage allowing red blood cells to leak into tick hemolymph. On the basis of the CD147 receptor being linked to tissue growth regulation in mammals, we speculate that silencing of the AamCD147 receptor blocked progression of the tick intermolt growth, a process that precedes tick engorgement and their spontaneous detachment of from the host to end feeding. The results are discussed in context of advances in tick molecular physiology.     

Feeding electrogram studies on the African cattle brown ear tick Rhipicephalus appendiculatus: evidence for an antifeeding effect of tick resistant serum

Abstract. Feeding behaviour of partially engorged Rhipicephalus appendiculatus (Neumann) (Acari: Ixodidae) on rabbit serum held in capillary tubes and placed over the tick mouthparts was studied using the feeding electrogram technique with simultaneous macro video photography. Correlation of electrical events with fluid movement in the vicinity of the tick's mouthparts and the capillary meniscus, permitted the characterization of an orderly sequence of signals, termed the 'Feeding Complex', associated with highest weight gains. This complex consisted of a 3–8 Hz fast-sucking waveform typically lasting 4–5 min, a sharp drop in potential at salivation, and rest lasting 1 or 2 min where no waveform or fluid movements occur. Very high impedence recordings from within the tick capitulum indicate that the fast-sucking waveform coincides with bursts of potentials corresponding to contraction of the pharyngeal dilator muscles, whereas during rest a tonic series of spikes signifies that the floor of the salivarium is actively lowered. Feeding electrograms of ticks fed on serum from tick-resistant rabbits showed significantly fewer feeding complexes. The weight gains achieved by these ticks were reduced correspondingly. This suggests that some of the humoral effectors of immunity have an antifeedant effect on this unusual parasite of rabbits.     

Proteomic Analysis of Cattle Tick Rhipicephalus (Boophilus) microplus Saliva: A Comparison between Partially and Fully Engorged Females

The cattle tick Rhipicephalus (Boophilus) microplus is one of the most harmful parasites affecting bovines. Similarly to other hematophagous ectoparasites, R. microplus saliva contains a collection of bioactive compounds that inhibit host defenses against tick feeding activity. Thus, the study of tick salivary components offers opportunities for the development of immunological based tick control methods and medicinal applications. So far, only a few proteins have been identified in cattle tick saliva. The aim of this work was to identify proteins present in R. microplus female tick saliva at different feeding stages. Proteomic analysis of R. microplus saliva allowed identifying peptides corresponding to 187 and 68 tick and bovine proteins, respectively. Our data confirm that (i) R. microplus saliva is complex, and (ii) that there are remarkable differences in saliva composition between partially engorged and fully engorged female ticks. R. microplus saliva is rich mainly in (i) hemelipoproteins and other transporter proteins, (ii) secreted cross-tick species conserved proteins, (iii) lipocalins, (iv) peptidase inhibitors, (v) antimicrobial peptides, (vii) glycine-rich proteins, (viii) housekeeping proteins and (ix) host proteins. This investigation represents the first proteomic study about R. microplus saliva, and reports the most comprehensive Ixodidae tick saliva proteome published to date. Our results improve the understanding of tick salivary modulators of host defense to tick feeding, and provide novel information on the tick-host relationship.     

Factors influencing the toxicity of salivary gland extracts of Ixodes holocyclus Neumann

A bioassay using mice was developed to compare the toxin content of extracts of salivary glands of I. holocyclus at various stages of feeding. The quantity of toxin increased rapidly from the third day of feeding. Toxin production continued and increased in ticks removed after 3–5 days on mice and held at 30°C at 92% RH for 24 h, whereas no toxin was detected in the salivary glands of ticks fed for 3 days and treated similarly. It is suggested that major physiological changes occur in the salivary glands of I. holocyclus on the third day, which once stimulated continue independently of feeding. Toxin production in ticks was not suppressed by passively immunizing mice with anti-tick toxin but was in ticks fed upon hosts with a previous experience of tick feeding.Thus, to obtain salivary glands containing high concentrations of toxin for chemical analysis, it is necessary for salivary glands to develop 5 days from the initial attachment of the tick to a host with no previous experience of tick feeding. This can be achieved by passively immunizing mice against toxin, thus enabling the tick to feed 5 days without killing the mouse or by keeping the tick for 24 h at 30°C at 92% RH following the death of the mouse on the fourth day.     

Prevalence,Diversity, and Load of Borrelia species in Ticks That Have Fed on Humans in Regions of Sweden and ?land Islands,Finland with Different Lyme Borreliosis Incidences

The incidence of Lyme borreliosis (LB) in a region may reflect the prevalence of Borrelia in the tick population. Our aim was to investigate if regions with different LB incidences can be distinguished by studying the prevalence and diversity of Borrelia species in their respective tick populations. The Borrelia load in a feeding tick increases with the duration of feeding, which may facilitate a transmission of Borrelia Spirochetes from tick to host. Therefore, we also wanted to investigate how the Borrelia load in ticks that have fed on humans varies with the duration of tick feeding. During 2008 and 2009, ticks that had bitten humans were collected from four regions of Sweden and Finland, regions with expected differences in LB incidence. The duration of tick feeding was estimated and Borrelia were detected and quantified by a quantitative PCR assay followed by species determination. Out of the 2,154 Ixodes ricinus ticks analyzed, 26% were infected with Borrelia and seven species were identified. B. spielmanii was detected for the first time in the regions. The tick populations collected from the four regions exhibited only minor differences in both prevalence and diversity of Borrelia species, indicating that these variables alone cannot explain the regions’ different LB incidences. The number of Borrelia cells in the infected ticks ranged from fewer than ten to more than a million. We also found a lower number of Borrelia cells in adult female ticks that had fed for more than 36 hours, compared to the number of Borrelia cells found in adult female ticks that had fed for less than 36 hours.     

Hemalin, a thrombin inhibitor isolated from a midgut cDNA library from the hard tick Haemaphysalis longicornis

A full-length sequence of a thrombin inhibitor (designated as hemalin) from the midgut of parthenogenetic Haemaphysalis longicornis has been identified. Sequence analysis shows that this gene belongs to the Kunitz-type family, containing two Kunitz domains with high homology to boophilin, the thrombin inhibitor from Rhipicephalus (Boophilus) microplus. The recombinant protein expressed in insect cells delayed bovine plasma clotting time and inhibited both thrombin-induced fibrinogen clotting and platelet aggregation. A 20-kDa protein was detected from the midgut lysate with antiserum against recombinant hemalin. The gene is expressed at all stages of the tick except for the egg stage, and hemalin mRNA mainly in the midgut of the female adult tick. Real-time PCR analysis shows that this gene has a distinctly high expression level in the rapid bloodsucking period of the larvae, nymphs, and adults. Disruption of the hemalin gene by RNA interference led to a 2-day extension of the tick blood feeding period, and 27.7% of the RNA-treated ticks did not successfully complete the blood feeding. These findings indicate that the newly identified thrombin inhibitor from the midgut of H. longicornis might play an important role in tick blood feeding.     

Feeding electrograms and fluid uptake measurements of cattle tick Boophilus microplus attached on artificial membranes

Waladde S. M., Kemp D. H. and Rice M. J. 1979. Feeding electrograms and fluid uptake measurements of cattle tick Boophilus microplus attached on artificial membranes. International Journal for Parasitology9: 89–95. Newly moulted adult females of the cattle tick Boophilus microplus readily attach on a modified Baudruche membrane. Apparatus design permits the media offered below the membrane to be kept at 37°C and to be changed easily. Patterns of feeding activity were recorded within 2 h of placing the ticks on the membrane by monitoring the changes in electrical resistance between a tick and the medium with a high input impedance electric circuit. Differences in patterns of sucking and salivation were related to the chemical composition of media presented below the membrane. These observations suggest that the newly discovered cheliceral taste receptors of B. microplus are able to mediate changes in feeding patterns in response to stimulation by different chemical solutions in the feeding lesion. Incorporation of phosphorus-32 into the medium allows the volume ingested by ticks to be measured. The techniques described here facilitate the study of host factors that influence attachment, engorgement and detachment of the cattle tick.     

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.     

Amblyomma maculatum Feeding Augments Rickettsia parkeri Infection in a Rhesus Macaque Model: A Pilot Study

Rickettsia parkeri is an emerging eschar-causing human pathogen in the spotted fever group of Rickettsia and is transmitted by the Gulf coast tick, Amblyomma maculatum. Tick saliva has been shown to alter both the cellular and humoral components of the innate and adaptive immune systems. However, the effect of this immunomodulation on Rickettsia transmission and pathology in an immunocompetent vertebrate host has not been fully examined. We hypothesize that, by modifying the host immune response, tick feeding enhances infection and pathology of pathogenic spotted fever group Rickettsia sp. In order to assess this interaction in vivo, a pilot study was conducted using five rhesus macaques that were divided into three groups. One group was intradermally inoculated with low passage R. parkeri (Portsmouth strain) alone (n = 2) and another group was inoculated during infestation by adult, R. parkeri-free A. maculatum (n = 2). The final macaque was infested with ticks alone (tick feeding control group). Blood, lymph node and skin biopsies were collected at several time points post-inoculation/infestation to assess pathology and quantify rickettsial DNA. As opposed to the tick-only animal, all Rickettsia-inoculated macaques developed inflammatory leukograms, elevated C-reactive protein concentrations, and elevated TH1 (interferon-γ, interleukin-15) and acute phase inflammatory cytokines (interleukin-6) post-inoculation, with greater neutrophilia and interleukin-6 concentrations in the tick plus R. parkeri group. While eschars formed at all R. parkeri inoculation sites, larger and slower healing eschars were observed in the tick feeding plus R. parkeri group. Furthermore, dissemination of R. parkeri to draining lymph nodes early in infection and increased persistence at the inoculation site were observed in the tick plus R. parkeri group. This study indicates that rhesus macaques can be used to model R. parkeri rickettsiosis, and suggests that immunomodulatory factors introduced during tick feeding may enhance the pathogenicity of spotted fever group Rickettsia.     

The Mitochondrial Genomes of Nuttalliella namaqua (Ixodoidea: Nuttalliellidae) and Argas africolumbae (Ixodoidae: Argasidae): Estimation of Divergence Dates for the Major Tick Lineages and Reconstruction of Ancestral Blood-Feeding Characters

Ixodida are composed of hard (Ixodidae), soft (Argasidae) and the monotypic Nuttalliellidae (Nuttalliella namaqua) tick families. Nuclear 18S rRNA analysis suggested that N. namaqua was the closest extant relative to the last common ancestral tick lineage. The mitochondrial genomes of N. namaqua and Argas africolumbae were determined using next generation sequencing and de novo assembly to investigate this further. The latter was included since previous estimates on the divergence times of argasids lacked data for this major genus. Mitochondrial gene order for both was identical to that of the Argasidae and Prostriata. Bayesian analysis of the COI, Cytb, ND1, ND2 and ND4 genes confirmed the monophyly of ticks, the basal position of N. namaqua to the other tick families and the accepted systematic relationships of the other tick genera. Molecular clock estimates were derived for the divergence of the major tick lineages and supported previous estimates on the origins of ticks in the Carboniferous. N. namaqua larvae fed successfully on lizards and mice in a prolonged manner similar to many argasids and all ixodids. Excess blood meal-derived water was secreted via the salivary glands, similar to ixodids. We propose that this prolonged larval feeding style eventually gave rise to the long feeding periods that typify the single larval, nymphal and adult stages of ixodid ticks and the associated secretion of water via the salivary glands. Ancestral reconstruction of characters involved in blood-feeding indicates that most of the characteristics unique to either hard or soft tick families were present in the ancestral tick lineage.     

Rhipicephalus sanguineus: sequential histopathology at the host-arthropod interface

The reaction beneath the mouth parts of an adult, female Rhipicephalus sanguineus attached to a dog develops progressively over 4–5 days. The cement substance is confined to the external surface of the epidermis and does not form any organized structure in the dermis. The hypostome is imbedded in the cement substance and does not penetrate the epidermis. The cheliceral shafts are at the epidermal-dermal junction and do not extend into the dermis to any degree. Thus, it is the adhesive quality of the cement for the dog's skin that holds this tick firmly attached to its host.Edema of the epidermis appears 24 hr after attachment of the tick; the dermal infiltrate becomes prominent from 24 hr after attachment onward and initially consists of polymorphonuclear leukocytes. This cell type predominates until the tick has detached. Rupture of lymphatics occurs and infiltrated cells can be found entering these open channels. Degranulation of mast cells is associated with polymorphonuclear leukocytic infiltration. A cavity develops in the dermis progressively over the period of tick attachment and feeding. This cavity appears during the first 24 hr and its full development depends upon leukocyte infiltration and the feeding activity of the tick. During the healing phase, macrophages, lymphocytes and fibroblasts gradually replace the polymorphonuclear leukocytes. The dermis is still hypercellular in the area of former attachment 2 wk after a female tick has detached fully engorged. The dog does not appear to develop resistance to the tick's engorgment even after 2 yr of intermittent exposure, nor does the host's reaction hinder the tick's engorgment. A dog never before exposed to arthropods of any kind reacted, histologically, to tick exposure with a very similar infiltration of polymorphonuclear leukocytes as seen in dogs repeatedly exposed. It is suggested that the inflammatory response by the host to the feeding tick may play an important role in the development and spread of infectious agents transmitted by this pool feeding arthropod.