Tick Ornithodoros Savignyi Research Articles

Molecular basis of anticoagulant and anticomplement activity of the tick salivary protein Salp14 and its homologs

During feeding, a tick's mouthpart penetrates the host's skin and damages tissues and small blood vessels, triggering the extrinsic coagulation and lectin complement pathways. To elude these defense mechanisms, ticks secrete multiple anticoagulant proteins and complement system inhibitors in their saliva. Here, we characterized the inhibitory activities of the homologous tick salivary proteins tick salivary lectin pathway inhibitor, Salp14, and Salp9Pac from Ixodesscapularis in the coagulation cascade and the lectin complement pathway. All three proteins inhibited binding of mannan-binding lectin to the polysaccharide mannan, preventing the activation of the lectin complement pathway. In contrast, only Salp14 showed an appreciable effect on coagulation by prolonging the lag time of thrombin generation. We found that the anticoagulant properties of Salp14 are governed by its basic tail region, which resembles the C terminus of tissue factor pathway inhibitor alpha and blocks the assembly and/or activity of the prothrombinase complex in the same way. Moreover, the Salp14 protein tail contributes to the inhibition of the lectin complement pathway via interaction with mannan binding lectin–associated serine proteases. Furthermore, we identified BaSO4-adsorbing protein 1 isolated from the tick Ornithodoros savignyi as a distant homolog of tick salivary lectin pathway inhibitor/Salp14 proteins and showed that it inhibits the lectin complement pathway but not coagulation. The structure of BaSO4-adsorbing protein 1, solved here using NMR spectroscopy, indicated that this protein adopts a noncanonical epidermal growth factor domain–like structural fold, the first such report for tick salivary proteins. These data support a mechanism by which tick saliva proteins simultaneously inhibit both the host coagulation cascade and the lectin complement pathway.

Efficacy and safety of ethanolic Curcuma longa extract as a treatment for sand tampan ticks in a rabbit model.

Background and Aim:The soft tick Ornithodoros savignyi is distributed throughout Africa, including Egypt. It primarily attacks camels, cattle, donkeys, and cows; and rarely affects humans. This study evaluated the acaricidal efficacy of ethanolic Curcuma longa extract (Turmeric) on the second nymphs of O. savignyi and then investigated the safety of this herb in rabbits.Materials and Methods:The nymphs were immersed in 10, 5, 2.5, 1.25, and 0.625 mg/ml ethanolic C. longa extract. An additional group was immersed in ethanol as a control. On the 1st, 7th, and 15th-day post-treatment, the mortality percentages, LC50, and LC95 were calculated. The ticks exposed to 10mg/ml ethanol C. longa extract were investigated by scanning electron microscopy (SEM). Three male New Zealand White rabbits were orally administered 2ml (two doses) of 10mg/ml ethanolic C. longa extract, and another three rabbits were orally given two doses of 2ml of absolute ethanol as a negative control. Histopathological examination of the kidney and liver hematology and the kidney and liver function was performed. Chemical analysis of the extract was determined by gas chromatography-mass spectrometry (GC-MS).Results:The LC50 and LC95 were 1.31 and 15.07, 1.07 and 8.56, and 0.81 and 6.97mg/ml on the 1st, 7th, and 15thday, respectively. SEM revealed that mamillae and spots on the surfaces of the treated ticks were not discriminating except for some clefts on the surfaces. The histological examination, blood profile, and biochemical analyses revealed no significant differences between the treated and untreated rabbits (p>0.05). GC/MS analysis revealed 50 compounds, and curcumene and tumerone were found to be the major constituents of this ethanolic extract.Conclusion:The ethanolic C. longa extract produced a strong acaricidal effect on the second nymph of O. savignyi, and it was safe to use in rabbits.

Antimicrobial function of short amidated peptide fragments from the tick-derived OsDef2 defensin.

Previously Os, a 22 amino acid sequence of a defensin from the soft tick Ornithodoros savignyi, was found to kill Gram-positive and Gram-negative bacteria at low micromolar concentrations. In this study, we evaluated synthetic peptide analogues of Os for antibacterial activity with an aim to identify minimalized active peptide sequences and in so doing obtain a better understanding of the structural requirements for activity. Out of eight partially overlapping sequences of 10 to 12 residues, only Os(3-12) and Os(11-22) exhibit activity when screened against Gram-positive and Gram-negative bacteria. Carboxyamidation of both peptides increased membrane-mediated activity, although carboxyamidation of Os(11-22) negatively impacted on activity against Staphylococcus aureus. The amidated peptides, Os(3-12)NH2 and Os(11-22)NH2 , have minimum bactericidal concentrations of 3.3μM against Escherichia coli. Killing was reached within 10minutes for Os(3-12)NH2 and only during the second hour for Os(11-22)NH2 . In an E. coli membrane liposome system, both Os and Os(3-12)NH2 were identified as membrane disrupting while Os(11-22)NH2 was less active, indicating that in addition to membrane permeabilization, other targets may be involved in bacterial killing. In contrast to Os, the membrane disruptive effect of Os(3-12)NH2 did not diminish in the presence of salt. Neither Os nor its amidated derivatives caused human erythrocyte haemolysis. The contrasting killing kinetics and effects of amidation together with structural and liposome leakage data suggest that the 3-12 fragment relies on a membrane disruptive mechanism while the 11-22 fragment involves additional target mechanisms. The salt-resistant potency of Os(3-12)NH2 identifies it as a promising candidate for further development.

Evaluation of vaccine candidates purified from the adult ticks of Ornithodoros savignyi (Acari: Argasidae) and Hyalomma dromedarii (Acari: Ixodidae) against tick infestations.

Ticks cause anemia, toxicosis, growth delay, and transmit infectious diseases in animals and humans. The current study aimed to evaluate the immunoprophylactic properties of two vaccine candidates to develop vaccine against tick infestations. These two vaccine candidates were specific fraction from the adults of the soft tick Ornithodoros savignyi and cross-reactive fraction from the adults of the hard tick Hyalomma dromedarii. Both specific and cross-reactive fractions were isolated by Cyanogen Bromide-activated Sepharose-4B affinity column chromatography. Both candidates proved their cross-reactivity by enzyme linked immunosorbent assay and Western blot. Characterization of the two vaccines by SDS-PAGE showed that the O. savignyi specific fraction consists of four bands; 97, 85, 66 and 11.5kDa compared with nine bands associated with its crude antigen (196-11.5kDa). The H. dromedarii cross-reactive vaccine candidate consists of three bands; 97, 66 and 45kDa compared to eight bands of its crude antigen (196-21kDa). Two common bands of 97 and 66kDa between two candidates showed immunogenic cross-reactivity with the developed antisera of both infestations by Western blot. Immunization of rabbits intramuscularly with two doses of the fractions separately (40µg/kg) led to immunological and parasitological changes. Immunologically; the level of immunoglobulins in vaccinated rabbits increased significantly compared with control infested non-vaccinated rabbits. These immunoglobulins are probably responsible for the protective effect of both candidates. Parasitologically, immunized rabbits showed protection against infestation by adult ticks as proved by significant feeding rejection percentage and significant reduction in egg and engorgement weights of H. dromedarii. While insignificant protection was observed against O. savignyi ticks infestation in feeding rejection and reduction in engorgement weight. In conclusion, this study suggests promising immunoprophylactic potentials of the purified fractions against tick infestations in rabbits through induction of IgG responses. The protective effect of both vaccine candidates deserves further evaluation in other hosts and against other tick infestations.

Anti-inflammatory and anti-endotoxin properties of peptides derived from the carboxy-terminal region of a defensin from the tick Ornithodoros savignyi.

Antimicrobial peptides are small cationic peptides that possess a large spectrum of bioactivities, including antimicrobial, anti-inflammatory and antioxidant activities. Several antimicrobial peptides are known to inhibit lipopolysaccharide (LPS)-induced inflammation in vitro and to protect animals from sepsis. In this study, the cellular anti-inflammatory and anti-endotoxin activities of Os and Os-C, peptides derived from the carboxy-terminal of a tick defensin, were investigated. Both Os and Os-C were found to bind LPS in vitro, albeit to a lesser extent than polymyxin B and melittin, known endotoxin-binding peptides. Binding to LPS was found to reduce the bactericidal activity of Os and Os-C against Escherichia coli confirming the affinity of both peptides for LPS. At a concentration of 25 µM, the nitric oxide (NO) scavenging activity of Os was higher than glutathione, a known NO scavenger. In contrast, Os-C showed no scavenging activity. Os and Os-C inhibited LPS/IFN-γ induced NO and TNF-α production in RAW 264.7 cells in a concentration-dependent manner, with no cellular toxicity even at a concentration of 100 µM. Although inhibition of NO and TNF-α secretion was more pronounced for melittin and polymyxin B, significant cytotoxicity was observed at concentrations of 1.56 µM and 25 µM for melittin and polymyxin B, respectively. In addition, Os, Os-C and glutathione protected RAW 264.7 cells from oxidative damage at concentrations as low as 25 µM. This study identified that besides previously reported antibacterial activity of Os and Os-C, both peptides have in addition anti-inflammatory and anti-endotoxin properties.

Exon?intron structure of outlier tick lipocalins indicate a monophyletic origin within the larger lipocalin family

All tick proteins assigned to the lipocalin family lack the structural conserved regions (SCRs) that are characteristic of the kernel lipocalins and can thus be classified as outliers. These tick proteins have been assigned to the tick lipocalin family based on database searches that indicated homology between tick sequences and the fact that the histamine binding protein (HBP2) from the hard tick Rhipicephalus appendiculatus (Ixodidae) shows structural similarity to the lipocalin fold. Sequence identity between kernel and outlier lipocalins falls below 20% and the question raised is whether the outlier and kernel lipocalins are truly homologous. More specifically in the case of the tick lipocalins, whether their structural fold is derived from the lipocalin fold or whether convergent evolution resulted in the generation of the basic lipocalin-like fold which consists of an eight stranded continuous anti-parallel β-barrel terminated by a C-terminal α-helix that lies parallel to the barrel. The current study determined the gene structure for HBP2 and TSGP1, TSGP2 and TSGP4, lipocalins identified from the soft tick Ornithodoros savignyi (Argasidae). All tick lipocalins have four introns (A–D) with conserved positions and phases within the tick lipocalin sequence alignment. The positions and phase information are also conserved with regard to the rest of the lipocalin family. Phylogenetic analysis using this information shows conclusively that tick lipocalins are evolutionary related to the rest of the lipocalin family. Tick lipocalins are grouped within a monophyletic clade that indicates a monophyletic origin within the tick lineage and also group with the other arthropod lipocalins in a larger clade. Phylogenetic analysis of sequence alignments based on conserved secondary structure of the lipocalin fold support the conclusions from the gene structure trees. These results indicate that exon–intron arrangement can be useful for the inclusion of outlier lipocalins within the larger lipocalin family.

Exon–intron structure of outlier tick lipocalins indicate a monophyletic origin within the larger lipocalin family

All tick proteins assigned to the lipocalin family lack the structural conserved regions (SCRs) that are characteristic of the kernel lipocalins and can thus be classified as outliers. These tick proteins have been assigned to the tick lipocalin family based on database searches that indicated homology between tick sequences and the fact that the histamine binding protein (HBP2) from the hard tick Rhipicephalus appendiculatus (Ixodidae) shows structural similarity to the lipocalin fold. Sequence identity between kernel and outlier lipocalins falls below 20% and the question raised is whether the outlier and kernel lipocalins are truly homologous. More specifically in the case of the tick lipocalins, whether their structural fold is derived from the lipocalin fold or whether convergent evolution resulted in the generation of the basic lipocalin-like fold which consists of an eight stranded continuous anti-parallel β-barrel terminated by a C-terminal α-helix that lies parallel to the barrel. The current study determined the gene structure for HBP2 and TSGP1, TSGP2 and TSGP4, lipocalins identified from the soft tick Ornithodoros savignyi (Argasidae). All tick lipocalins have four introns (A–D) with conserved positions and phases within the tick lipocalin sequence alignment. The positions and phase information are also conserved with regard to the rest of the lipocalin family. Phylogenetic analysis using this information shows conclusively that tick lipocalins are evolutionary related to the rest of the lipocalin family. Tick lipocalins are grouped within a monophyletic clade that indicates a monophyletic origin within the tick lineage and also group with the other arthropod lipocalins in a larger clade. Phylogenetic analysis of sequence alignments based on conserved secondary structure of the lipocalin fold support the conclusions from the gene structure trees. These results indicate that exon–intron arrangement can be useful for the inclusion of outlier lipocalins within the larger lipocalin family.

A reassessment of argasid tick salivary gland ultrastructure from an immuno-cytochemical perspective

Previous morphological and histochemical studies of argasid tick salivary glands indicated that they were less complex than ixodid salivary glands, with only three granular cell types. The present study shows that there exist at least four different granular cell types in the salivary glands of the argasid tick Ornithodoros savignyi, based on immuno-localization of the anti-hemostatic factors, apyrase and savignygrin. Both anti-hemostatic factors were localized to dense core granule type 'a' and to granule type 'b', that shares a similar homogenous morphology with non-labeled granule type 'd'. Furthermore, the major tick salivary gland proteins (TSGPs), previously implicated in granule biogenesis, were localized to all the granular cell types. This indicates that granular cell types with different morphologies can express the same proteins, while cell types that show similar morphologies may not express the same proteins. Argasid tick salivary glands seem to be more complex than previously thought and might not be amenable to morphological classification alone. Alternative classification methodologies that rely on physical expression patterns of the salivary gland proteome might be more reliable as markers for a specific granular cell type.

Savignygrin, a Platelet Aggregation Inhibitor from the Soft TickOrnithodoros savignyi, Presents the RGD Integrin Recognition Motif on the Kunitz-BPTI Fold

Savignygrin, a platelet aggregation inhibitor that possesses the RGD integrin recognition motif, has been purified from the soft tick Ornithodoros savignyi. Two isoforms with similar biological activities differ because of R52G and N60G in their amino acid sequences, indicating a recent gene duplication event. Platelet aggregation induced by ADP (IC50, 130 nm), collagen, the thrombin receptor-activating peptide, and epinephrine was inhibited, although platelets were activated and underwent a shape change. The binding of alpha-CD41 (P2) to platelets, the binding of purified alpha(IIb)beta3 to fibrinogen, and the adhesion of platelets to fibrinogen was inhibited, indicating a targeting of the fibrinogen receptor. In contrast, the adhesion of osteosarcoma cells that express the integrin alpha(v)beta3 to vitronectin or fibrinogen was not inhibited, indicating the specificity of savignygrin toward alpha(IIb)beta3. Savignygrin shows sequence identity to disagregin, a platelet aggregation inhibitor from the tick Ornithodoros moubata that lacks an RGD motif. The cysteine arrangement of savignygrin is similar to that of the bovine pancreatic trypsin inhibitor family of serine protease inhibitors. A homology model based on the structure of the tick anticoagulant peptide indicates that the RGD motif is presented on the substrate-binding loop of the canonical BPTI inhibitors. However, savignygrin did not inhibit the serine proteases fXa, plasmin, thrombin, or trypsin. This is the first report of a platelet aggregation inhibitor that presents the RGD motif using the Kunitz-BPTI protein fold.

Amino acid sequence and structure modeling of savignin, a thrombin inhibitor from the tick, Ornithodoros savignyi

The full-length gene of savignin, a potent thrombin (E.C. 3.4.21.5) inhibitor from the tick Ornithodoros savignyi has been cloned and sequenced. Both 5′ and 3′ UTR’s, a signal peptide from the translated amino acid sequence and an unusual poly-adenylation signal (AATACA) has been identified. The translated protein sequence shows high identity (63%) with ornithodorin, the thrombin inhibitor from the tick, Ornithodoros moubata. Molecular modeling using the structure of ornithodorin as reference gave a structure with an RMSD of 0.25 Å for the full-length protein, 0.11 Å for the N-terminal BPTI-like domain and 0.11 Å for the C-terminal BPTI-like domain, indicating that maximum deviation occurs in the mobile bridge (0.18 Å) between the two domains. Docking of savignin to thrombin shows that the interaction is similar to the ornithodorin–thrombin complex. The N-terminal amino acid residues of savignin bind inside the active site cleft, while the C-terminal domain of savignin has a net negative electrostatic potential and interacts with the basic fibrinogen recognition exosite of thrombin through hydrogen bonds and hydrophobic interactions. These results correlate with kinetic data obtained, which showed that savignin is a competitive, slow, tight-binding inhibitor that requires thrombin’s fibrinogen-binding exo-site for optimal inhibition.

Disaggregation of aggregated platelets by apyrase from the tick, Ornithodoros savignyi (Acari: Argasidae).

Apyrase, secreted by ticks during feeding, is a platelet aggregation inhibitor that functions as a regulator of the host's hemostatic system. This present study concerns the disaggregation effect of salivary gland apyrase from the tick Ornithodoros savignyi. Secondarily aggregated platelets, disaggregated by apyrase, exhibited a reversal of shape from a spherical (aggregated) form to a discoid form, reminiscent of reversible aggregation at low ADP concentrations in citrated platelet-rich plasma. However, they showed a dilatory open canaliculary system and an absence of granules indicating disaggregation after degranulation had taken place. In contrast, disaggregation by the fibrin(ogen)olytic enzyme, plasmin, showed that platelets degranulated, but retained a spherical form with numerous extended pseudopods. While thrombin had no effect on aggregation or clotting of platelets disaggregated with plasmin, it did activate those platelets disaggregated with apyrase and clotted the plasma. This is the first study to describe the disaggregating effects of tick derived apyrase on aggregated platelets. It also shows that apyrase can disaggregate platelets even after secondary aggregation and degranulation of platelets has taken place. Platelet aggregation is one of the main barriers encountered by ticks during feeding and counteraction of this process by ticks is an important factor for successful feeding.

Savignin, a Potent Thrombin Inhibitor Isolated from the Salivary Glands of the Tick Ornithodoros savignyi (Acari: Argasidae)

Nienaber, J., Gaspar, A. R. M., and Neitz, A. W. H. 1999. Savignin, a potent thrombin inhibitor isolated from the salivary glands of the tick Ornithodoros savignyi (Acari: Argasidae). Experimental Parasitology93, 82–91. A thrombin (E.C. 3.4.21.5) inhibitor, savignin, was isolated from the salivary glands of Ornithodoros savignyi by a combination of size exclusion, anion-exchange, and reversed-phase chromatography. The inhibitor has a molecular mass of 12,430.4 Da as determined by electrospray mass spectrometry. The behavior of savignin during anion-exchange chromatography indicated that it has an acidic pI. The available N-terminal sequence (residues 1–11) differed from that of ornithodorin with only one residue. Savignin inhibits thrombin-induced platelet aggregation, but has no effect on ADP- or collagen-induced aggregation. Kinetic studies indicated that savignin is a competitive, slow-, tight-binding inhibitor of α-thrombin (Ki = 4.89 ± 1.39 pM). Tight-binding kinetics showed that the inhibitor has a lower affinity for γ-thrombin (Ki = 22.3 ± 5.9 nM). Plasmin, factor Xa, and trypsin are not inhibited by savignin.

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 activitives 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.

Laboratory vector studies on six mosquito and one tick species with chikungunya virus

The tick Ornithodoros savignyi and the mosquitoes Culex horridus, Culex quinquefasciatus, Aedes fulgens, Ae. furcifer and Mansonia africana were tested for infection rates and ability to transmit chikungunya virus. O. savignyi and Cx quinquefasciatus did not become infected and Cx quinquefasciatus failed to transmit the virus between vervet monkeys, Cercopithecus aethiops. Only one of 17 Cx horridus feeding on a blood-virus mixture became infected which included infection of the salivary glands. Ae. fulgens had a high infection rate and transmitted the virus between Mystromys albicaudatus rodents. Ae. furcifer and Ma. africana both transmitted virus between vervet monkeys: the 50% infection threshold and the transmission rate were < 4·5 logs and 25% respectively for Ae. furcifer and c. 5·5 logs and 29% for Ma. africana. In a further test, Ae. furcifer transmitted virus from a monkey to hamsters at a transmission rate of 32%. Attempts to demonstrate transovarial transmission of the virus in Ae. aegypti and Ae. furcifer were unsuccessful. It is concluded that Ae. furcifer is fitted for its suspected role as epidemic vector and that Ma. africana could also act as an important epidemic vector in southern Africa.

Chemical composition of the coxal fluid of the argasid tick Ornithodoros savignyi.

A study was made of the chemical composition of the coxal fluid of the argasid tick Ornithodoros savignyi . The main compounds determined were certain electrolytes, amino acids, proteins, nucleic acids, carbohydrates and lipids. In addition the electrolyte content of the hemolymph was measured. Substantial amounts of sodium, potassium, chloride and bicarbonate were eliminated by the coxal glands, whereas small quantities of calcium, magnesium and phosphates were eliminated. In the hemolymph, these electrolytes occurred in a ratio different from that in the coxal fluid. Several amino acids were detected in the coxal fluid. In contrast to the amino acids, protein and certain other non-protcin nitrogenous compounds were eliminated in comparatively large quantities. Lipids of all classeswere likewise eliminated in the coxal fluid, although to varied extents with phospholipids constituting the major portion. Of the carbohydrates studied, only glucose was eliminated in substantial quantities. Of particular interest was the detection of a small amount of sucrose. The present study confirms that the coxal organs are osmoregulatory organs which eliminate a wide range of compounds.

A rickettsialike microorganism in the tick Ornithodoros savignyi; Observations on its structure and distribution in the tissues of the tick

A rickettsialike microorganism is described, for the first time, from the tick Ornithodoros savignyi collected from camels in Egypt. The microorganism is found in the cells of the anterior half of the Malpighian tubules, except those of the anteriormost region of the organ, in both male and female ticks, and in the oocytes. It is not observed in the male gonads. In smears of infected organs stained with Giemsa or Macchiavello's stain, the microorganism appears as threads and chains of variable lengths, occurring either singly or in bundles, as well as in coccoid and short-rod forms. Light and electron microscopy of the microorganism in infected tissues show close similarities in appearance and fine structure to certain Rickettsia and to other rickettsialike symbiotes naturally occurring in ticks and other arthropods.

West Nile virus infection in arthropods.

Summary 1. Mosquitoes, ticks, houseflies, and probably human body lice were found to be susceptible to parenteral infection with West Nile virus. Chicken mites, bedbugs, rat fleas, hippoboscid flies, and cockroaches proved to be refractory. 2. Culex univittatus, C. antennatus, and C. pipiens were infected readily when blood having a virus titer of 4.5 was ingested. A titer of 2.5 was sufficient to infect many individuals. 3. C. pipiens and C. univittatus became infected and were capable of transmitting the virus when kept at mean daily temperatures of 12–23°C. 4. C. univittatus transmitted the virus as soon as 5 days after the infective blood meal and for at least 33 days thereafter at 28–32°C. 5. The tick Ornithodoros savignyi, when infected parenterally, transmitted the virus while biting infant mice, and the virus was isolated from the coxal fluid. This tick was also infected by feeding on the blood of infected mice, but failed to transmit the virus when infected in this manner. Ornithodoros erraticus and Argas persicus showed evidence of infection but did not transmit the virus.