Ornithodoros Moubata Research Articles

Double blockade of CD14 and complement C5 abolishes the cytokine storm and improves morbidity and survival in polymicrobial sepsis in mice.

Sepsis and septic shock, caused by an excessive systemic host-inflammatory response, are associated with high morbidity and mortality. The complement system and TLRs provide important pattern recognition receptors initiating the cytokine storm by extensive cross-talk. We hypothesized that double blockade of complement C5 and the TLR coreceptor CD14 could improve survival of experimental polymicrobial sepsis. Mice undergoing cecal ligation and puncture (CLP)-induced sepsis were treated with neutralizing anti-CD14 Ab biG 53, complement C5 inhibitor coversin (Ornithodoros moubata C inhibitor), or a combination thereof. The inflammatory study (24-h observation) revealed statistically significant increases in 22 of 24 measured plasma biomarkers in the untreated CLP group, comprising 14 pro- and anti-inflammatory cytokines and 8 chemokines, growth factors, and granulocyte activation markers. Single CD14 or C5 blockade significantly inhibited 20 and 19 of the 22 biomarkers, respectively. Combined CD14 and C5 inhibition significantly reduced all 22 biomarkers (mean reduction 85%; range 54-95%) compared with the untreated CLP group. Double blockade was more potent than single treatment and was required to significantly inhibit IL-6 and CXCL1. Combined inhibition significantly reduced morbidity (motility and eyelid movement) and mortality measured over 10 d. In the positive control CLP group, median survival was 36 h (range 24-48 h). Combined treatment increased median survival to 96 h (range 24-240 h) (p = 0.001), whereas survival in the single-treatment groups was not significantly increased (median and range for anti-CD14 and anti-C5 treatment were 36 h [24-48 h] and 48 h [24-96 h]). Combined with standard intervention therapy, specific blockade of CD14 and C5 might represent a promising new therapeutic strategy for treatment of polymicrobial sepsis.

Inhibition of the Membrane Attack Complex of the Complement System Reduces Secondary Neuroaxonal Loss and Promotes Neurologic Recovery after Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) is the leading cause of disability and death in young adults. The secondary neuroinflammation and neuronal damage that follows the primary mechanical injury is an important cause of disability in affected people. The membrane attack complex (MAC) of the complement system is detected in the traumatized brain early after TBI; however, its role in the pathology and neurologic outcome of TBI has not yet been investigated. We generated a C6 antisense oligonucleotide that blocks MAC formation by inhibiting C6, and we compared its therapeutic effect to that of Ornithodoros moubata complement inhibitor (OmCI), a known inhibitor of C5 activation that blocks generation of the anaphylatoxin C5a and C5b, an essential component of MAC. Severe closed head injury in mice induced abundant MAC deposition in the brain. Treatment with C6 antisense reduced C6 synthesis (85%) and serum levels (90%), and inhibited MAC deposition in the injured brain (91-96%). Treatment also reduced accumulation of microglia/macrophages (50-88%), neuronal apoptosis, axonal loss and weight loss (54-93%), and enhanced neurologic performance (84-92%) compared with placebo-treated controls after injury. These data provide the first evidence, to our knowledge, that inhibition of MAC formation in otherwise complement-sufficient animals reduces neuropathology and promotes neurologic recovery after TBI. Given the importance of maintaining a functional complement opsonization system to fight infections, a critical complication in TBI patients, inhibition of the MAC should be considered to reduce posttraumatic neurologic damage. This work identifies a novel therapeutic target for TBI and will guide the development of new therapy for patients.

The novel isoxazoline ectoparasiticide fluralaner: Selective inhibition of arthropod γ-aminobutyric acid- and l-glutamate-gated chloride channels and insecticidal/acaricidal activity

Isoxazolines are a novel class of parasiticides that are potent inhibitors of γ-aminobutyric acid (GABA)-gated chloride channels (GABACls) and l-glutamate-gated chloride channels (GluCls). In this study, the effects of the isoxazoline drug fluralaner on insect and acarid GABACl (RDL) and GluCl and its parasiticidal potency were investigated. We report the identification and cDNA cloning of Rhipicephalus (R.) microplus RDL and GluCl genes, and their functional expression in Xenopus laevis oocytes. The generation of six clonal HEK293 cell lines expressing Rhipicephalus microplus RDL and GluCl, Ctenocephalides felis RDL-A285 and RDL-S285, as well as Drosophila melanogaster RDLCl-A302 and RDL-S302, combined with the development of a membrane potential fluorescence dye assay allowed the comparison of ion channel inhibition by fluralaner with that of established insecticides addressing RDL and GluCl as targets. In these assays fluralaner was several orders of magnitude more potent than picrotoxinin and dieldrin, and performed 5-236 fold better than fipronil on the arthropod RDLs, while a rat GABACl remained unaffected. Comparative studies showed that R. microplus RDL is 52-fold more sensitive than R. microplus GluCl to fluralaner inhibition, confirming that the GABA-gated chloride channel is the primary target of this new parasiticide. In agreement with the superior RDL on-target activity, fluralaner outperformed dieldrin and fipronil in insecticidal screens on cat fleas (Ctenocephalides felis), yellow fever mosquito larvae (Aedes aegypti) and sheep blowfly larvae (Lucilia cuprina), as well as in acaricidal screens on cattle tick (R. microplus) adult females, brown dog tick (Rhipicephalus sanguineus) adult females and Ornithodoros moubata nymphs. These findings highlight the potential of fluralaner as a novel ectoparasiticide.

Identification of the Immunogenic Outer Membrane Proteins of Relapsing Fever <i>Borrelia</i>

Borrelia duttonii, a causative agent of relapsing fever, is transmitted between the distinct microenvironments of the vector tick, Ornithodoros moubata, and a mammalian host. We identified the total and membrane fraction proteins of B. duttonii strain Ly isolated from a patient in Tanzania by using two-dimensional gel electrophoresis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The analyses of the total and membrane fractions from bacterial cultures incubated at 37°C identified 68 and 15 proteins, respectively. Since spirochaete clearance in mice is associated with an immunoglobulin M (IgM) and immunoglobulin G3 (IgG3)-mediated response, immunoblot analyses were used to identify the proteins reactive with IgM and IgG3 of gerbil serum against B. duttonii strain Ly. The outcome showed that six proteins (antigen p83/100, membrane-associated protein P66 (P66), flagellar filament outer layer protein, hypothetical protein BDU_412, vlp protein gamma subfamily (γ-Vlp) and flagellin (FlaB)) were identified against IgM, and four (antigen p83/100, P66, γ-Vlp and FlaB) of the six proteins also reacted with IgG3. It is believed that these proteins are immunodominant antigens for the host immune response. Some of these immunogenic proteins might be used as molecular diagnostic tools in the study of relapsing fever in Tanzania.

Susceptibility of tick cell lines to infection with Alkhumra haemorrhagic fever virus

Although Alkhumra haemorrhagic fever virus (AHFV) has been isolated from ticks, epidemiological data suggest that it is transmitted from livestock to humans by direct contact with animals or by mosquito bites, but not by ticks. This study was carried out to assess the ability of the virus to replicate in tick cells in vitro. AHFV was inoculated into cell lines derived from the hard ticks Hyalomma anatolicum (HAE/CTVM9) and Rhipicephalus appendiculatus (RAE/CTVM1) and the soft tick Ornithodoros moubata (OME/CTVM24). Inoculated cells were directly examined every week for 4 weeks by real-time reverse transcription PCR and by IFAT using polyclonal antibodies. AHFV RNA was detected in all three inoculated tick cell lines throughout the 4-week observation period at levels up to almost twice that of the inoculum, but none of them exhibited a cytopathic effect. AHFV antigen could be detected in all three cell lines by IFAT. Titration of tick cell culture suspension in LLC-MK2 cells yielded AHFV titres of 10(6.6) 50% tissue culture infective dose (TCID50)/ml for OME/CTVM24 and 10(5.5) TCID50/ml for RAE/CTVM1 cells after 4 weeks of culturing; no viable virus was detected in HAE/CTVM9 cells. This is the first description of propagation of AHFV in tick cells.

Combined Inhibition of Complement (C5) and CD14 Markedly Attenuates Inflammation, Thrombogenicity, and Hemodynamic Changes in Porcine Sepsis

Complement and the TLR family constitute two important branches of innate immunity. We previously showed attenuating effects on inflammation and thromogenicity by inhibiting the TLR coreceptor CD14 in porcine sepsis. In the present study, we explored the effect of the C5 and leukotriene B4 inhibitor Ornithodoros moubata complement inhibitor (OmCI; also known as coversin) alone and combined with anti-CD14 on the early inflammatory, hemostatic, and hemodynamic responses in porcine Escherichia coli-induced sepsis. Pigs were randomly allocated to negative controls (n = 6), positive controls (n = 8), intervention with OmCI (n = 8), or with OmCI and anti-CD14 (n = 8). OmCI ablated C5 activation and formation of the terminal complement complex and significantly decreased leukotriene B4 levels in septic pigs. Granulocyte tissue factor expression, formation of thrombin-antithrombin complexes (p < 0.001), and formation of TNF-α and IL-6 (p < 0.05) were efficiently inhibited by OmCI alone and abolished or strongly attenuated by the combination of OmCI and anti-CD14 (p < 0.001 for all). Additionally, the combined therapy attenuated the formation of plasminogen activator inhibitor-1 (p < 0.05), IL-1β, and IL-8, increased the formation of IL-10, and abolished the expression of wCD11R3 (CD11b) and the fall in neutrophil cell count (p < 0.001 for all). Finally, OmCI combined with anti-CD14 delayed increases in heart rate by 60 min (p < 0.05) and mean pulmonary artery pressure by 30 min (p < 0.01). Ex vivo studies confirmed the additional effect of combining anti-CD14 with OmCI. In conclusion, upstream inhibition of the key innate immunity molecules, C5 and CD14, is a potential broad-acting treatment regimen in sepsis as it efficiently attenuated inflammation and thrombogenicity and delayed hemodynamic changes.

An insight into the proteome of the saliva of the argasid tick Ornithodoros moubata reveals important differences in saliva protein composition between the sexes

Tick saliva contains pharmacologically active molecules that allow these parasites to obtain a blood meal from the host and facilitate host infection by tick-borne pathogens. Recent transcriptomic and proteomic analyses of the salivary glands of several tick species have provided data sets that are invaluable for a better understanding of tick sialomes and tick-host-pathogen relationships. Here we performed a proteomic study of the saliva from the argasid tick Ornithodoros moubata. Saliva samples from female and male specimens were analyzed separately by LC-MS/MS before and after their equalization to facilitate the identification of the less abundant proteins. We report the array of 193 proteins identified in the saliva of O. moubata showing: (i) the broad and complex composition of the saliva of this tick, in good agreement with the complexity of the argasid and ixodid sialomes described previously; (ii) a notable difference in the saliva proteomes of females and males, since only 10 of the proteins identified appeared to be shared by both sexes; and (iii) the presence in the salivary fluid of a wide range of proteins known to be housekeeping/intracellular, which could be secreted in unconventional ways, including exosome secretion.

Self-assembled Protein Arrays from anOrnithodoros moubataSalivary Gland Expression Library

Protein interactions play a critical role in the regulation of many biological events and their study in a high-throughput format has become a key area of proteomic research. Nucleid Acid Programmable Protein Arrays (NAPPA) technology allows the construction of protein arrays from cDNA expression libraries in high-throughput cell-free systems to study protein interaction and functions. Tick saliva contains antihemostatic, anti-inflammatory, and immunosuppressive proteins that counteract the host hemostatic, immune, and inflammatory responses allowing the ingestion of host blood and facilitating its infection by the tick-borne pathogens. Identification of such proteins and their functions could help in the selection of antigenic targets for the development of antitick and transmission-blocking vaccines. With that aim, we have prepared a cDNA expression library from the salivary glands of Ornithodoros moubata and subsequently produced a self-assembled protein microarray using 480 randomly selected clones from that library. The reproducibility of the array, its representativeness of the tick salivary protein repertoire, and the functionality of the in situ expressed proteins have been checked, demonstrating that it is a suitable tool for the identification and functional characterization of soft tick salivary molecules that interact with host proteins. Several clones in the array were shown to bind to human recombinant P-selectin. One of them was a likely secreted tick phospholipase A2, which may represent a potential new ligand for P-selectin. As these salivary molecules are likely involved in blood meal acquisition through the modulation of the host immune and hemostatic responses, this new high-throughput tool could open new avenues for development of new therapeutic agents and control strategies against ticks and tick-borne pathogens.

Cloning and characterization of a plasminogen-binding enolase from the saliva of the argasid tick Ornithodoros moubata

Significant amounts of enolase have recently been found in the saliva of the argasid tick Ornithodoros moubata, raising the question as to what the function of enolase in the tick–host interface is. Enolase is a multifunctional glycolytic enzyme known to act as a plasminogen receptor on cellular surfaces, promoting fibrinolysis and extracellular matrix degradation. Fibrinolysis could be important for ticks to dissolve clots that might be formed during feeding as well as to prevent clotting of the ingested blood meal in the tick midgut. Additionally, enolase-mediated extracellular matrix degradation could contribute to the tick feeding lesion. Moreover, previous observations suggested an additional antihaemostatic role for O. moubata enolase as a P-selectin antagonist ligand. Accordingly, the aim of the present study was to investigate the potential role of the O. moubata salivary enolase as a plasminogen receptor and P-selectin ligand, and to evaluate its potential as an antigen target for anti-O. moubata vaccines. The study included the cloning, sequencing and recombinant production of the O. moubata enolase, plasminogen binding and activation assays, P-selectin binding assays, animal immunization trials, and RNAi knockdown of the enolase gene. Here we confirmed that enolase is secreted to the saliva of the tick and provide convincing evidence for a role of this salivary enolase as a plasminogen receptor, most likely stimulating host fibrinolysis and maintaining blood fluidity during tick feeding. The RNAi experiments and immunization trials indicated that enolase could be also involved in the regulation of tick reproduction, suggesting new potential control strategies. Finally, the P-selectin binding experiments demonstrated that this enolase is not a P-selectin ligand.

Ornithodoros moubata Complement Inhibitor Is an Equally Effective C5 Inhibitor in Pigs and Humans

Experimental evidence suggests that C inhibition and more particularly combined inhibition of C and the TLR coreceptor CD14 may be of therapeutic benefit in sepsis and other inflammatory conditions. A barrier to the testing and further development of many inhibitors is that their activity is species specific. Pig is a relevant species for experimental models of human disease, and this study undertakes a comprehensive comparison of the inhibitory efficacy of the C5 inhibitor Ornithodoros moubata C inhibitor (OmCI) in human and porcine whole blood ex vivo models of Escherichia coli-induced sepsis. The effect of OmCI on complement activity in pigs undergoing E. coli sepsis was also examined. Porcine and human serum, and whole blood anticoagulated with lepirudin, was incubated with E. coli and the effect of OmCI investigated. The ex vivo results were virtually identical in pig and human. OmCI completely ablated the activity of all three C pathways at 0.64 μM. E. coli-induced C activation and expression of CD11b (wCD11R3 in the pig), was abolished ex vivo at 0.32 μM OmCI. Combining anti-CD14 and OmCI reduced the formation of IL-8 and TNF-α more potently than the single inhibitors. OmCI also efficiently bound E. coli-induced leukotriene B(4) in pig and human plasma. In support of our ex vivo findings, in vivo the activity of all C pathways was inhibited at 0.6 mg OmCI/kg pig. In conclusion, OmCI efficiently inhibited pig and human C activation, has accompanying anti-inflammatory effects and is a promising candidate inhibitor for further in vivo studies of sepsis.

Tick cell lines for study of Crimean-Congo hemorrhagic fever virus and other arboviruses.

Continuous cell lines derived from many of the vectors of tick-borne arboviruses of medical and veterinary importance are now available. Their role as tools in arbovirus research to date is reviewed and their potential application in studies of tick cell responses to virus infection is explored, by comparison with recent progress in understanding mosquito immunity to arbovirus infection. A preliminary study of propagation of the human pathogen Crimean-Congo hemorrhagic fever virus (CCHFV) in tick cell lines is reported; CCHFV replicated in seven cell lines derived from the ticks Hyalomma anatolicum (a known vector), Amblyomma variegatum, Rhipicephalus (Boophilus) decoloratus, Rhipicephalus (Boophilus) microplus, and Ixodes ricinus, but not in three cell lines derived from Rhipicephalus appendiculatus and Ornithodoros moubata. This indicates that tick cell lines can be used to study growth of CCHFV in arthropod cells and that there may be species-specific restriction in permissive CCHFV infection at the cellular level.

Ornithodoros moubata complement inhibitor (OmCI) is an equally effective C5 inhibitor in pig and human, and combined with CD14 inhibition, efficiently attenuate Escherichia coli-induced sepsis in pigs

Ornithodoros moubata complement inhibitor (OmCI) is an equally effective C5 inhibitor in pig and human, and combined with CD14 inhibition, efficiently attenuate Escherichia coli-induced sepsis in pigs

Fibrinogen-related proteins in ixodid ticks

BackgroundFibrinogen-related proteins with lectin activity are believed to be part of the tick innate immune system. Several fibrinogen-related proteins have been described and characterised mainly on the basis of their cDNA sequences while direct biochemical evidence is missing. One of them, the haemolymph lectin Dorin M from the tick Ornithodoros moubata was isolated and characterised in more depth.ResultsSeveral fibrinogen-related proteins were detected in the haemolymph of ixodid ticks Dermacentor marginatus, Rhipicephalus appendiculatus, R. pulchellus, and R. sanguineus. These proteins were recognised by sera directed against the tick lectin Dorin M and the haemagglutination activity of the ticks R. appendiculatus and D. marginatus. Cross-reactivity of the identified proteins with antibodies against the fibrinogen domain of the human ficolin was also shown. The carbohydrate-binding ability of tick haemolymph was confirmed by haemagglutination activity assays, and this activity was shown to be inhibited by neuraminic acid and sialylated glycoproteins as well as by N-acetylated hexosamines. The fibrinogen-related proteins were shown to be glycosylated and they were localised in salivary glands, midguts, and haemocytes of D. marginatus. Hemelipoglycoprotein was also recognised by sera directed against the fibrinogen-related proteins in all three Rhipicephalus species as well as in D. marginatus. However, this protein does not contain the fibrinogen domain and thus, the binding possibly results from the structure similarity between hemelipoglycoprotein and the fibrinogen domain.ConclusionsThe presence of fibrinogen-related proteins was shown in the haemolymph of four tick species in high abundance. Reactivity of antibodies directed against ficolin or fibrinogen-related proteins with proteins which do not contain the fibrinogen domain points out the importance of sequence analysis of the identified proteins in further studies. Previously observed expression of fibrinogen-related proteins in haemocytes together with the results of this study suggest involvement of fibrinogen-related proteins in tick immunity processes. Thus, they have potential as targets for anti-tick vaccines and as antimicrobial proteins in pharmacology. Research on fibrinogen-related proteins could reveal further details of tick innate immunity processes.

Tick pathogenicity, thermal tolerance and virus infection in Tolypocladium cylindrosporum

Tolypocladium cylindrosporum is a fungus which has been isolated from soil, from asymptomatic plants as an endophyte and has been shown to be pathogenic to several species of arthropods. The objective of the present work was to study a collection of T. cylindrosporum strains to evaluate the characteristics of this fungus as a bioacaricide. The pathogenicity of five different strains of T. cylindrosporum was tested against two tick species, Ornithodoros erraticus and Ornithodoros moubata. Both tick species were susceptible to all the fungal strains. Mortality was greater for O. erraticus and differed among the five developmental stages of the ticks tested and among the fungal treatments. Mean mortality rates were close to 60% for O. erraticus, similar to those reported for other entomopathogenic fungi used for this purpose. The responses of 11 different strains of the fungus to 22°C and 30°C were also studied. Significant differences in temperature tolerance occurred among the strains and growth inhibition was observed at 30°C. Several mycoviruses were found infecting 5 of the 11 strains. However, no clear relationship was found between the presence of viruses and fungal growth or pathogenicity.

Assessment of interactions between African swine fever virus, bushpigs (Potamochoerus larvatus), Ornithodoros ticks and domestic pigs in north-western Madagascar

Since its introduction in Madagascar in 1998, African swine fever (ASF) has severely affected national pig production and persists as a common disease in that country. Two of its natural hosts in the African continent, the bushpig (Potamochoerus larvatus) and tick vectors of the Ornithodoros moubata complex, are reported in west and central regions of the island. However, their role in the maintenance and transmission of the virus has been insufficiently studied. In this work, we tried to assess their potential role in the epidemiology of the disease in Madagascar, by assessing the levels of interaction between (i) ASF virus (ASFV) and bushpigs and (ii) between soft ticks and domestic and wild suids in north-western Madagascar. Twenty-seven sera and 35 tissue samples from bushpigs were collected and analysed for the presence of anti-ASF antibodies and viral DNA. In addition, the sera from 27 bushpigs and 126 domestic pigs were analysed with an ELISA test for the detection of antibodies against salivary antigens from Ornithodoros ticks. No circulation of ASFV or anti-ASFV antibodies nor anti-tick antibodies were detected in bushpigs. However, seven of the domestic pig sera (5.6% of the total sample population) were antibody positive for O.moubata antigens. The probability of freedom from ASFV in the bushpig population using Bayesian statistical methods ranged between 73% and 84%. The probabilities of absence of anti-tick antibodies in domestic and wild pigs were estimated at 63% and 71%, respectively. These preliminary results suggest that bushpigs are unlikely to play a significant role in the maintenance and transmission of ASFV in Madagascar. Nevertheless, further ASFV surveys are needed on that species to confirm this assumption. In addition, the presence of antibodies against O.moubata in domestic pigs suggests that soft ticks may be able to maintain ASFV within a domestic pig cycle in areas of Madagascar where they remain present.

Hemelipoglycoprotein from the ornate sheep tick, Dermacentor marginatus: structural and functional characterization

BackgroundTick carrier proteins are able to bind, transport, and store host-blood heme, and thus they function also as antioxidants. Nevertheless, the role of carrier proteins in ticks is not fully understood. Some of them are found also in tick males which do not feed on hosts to such an extent such as females (there are differences in male feeding in different tick species) and thus they are not dealing with such an excess of heme; some of the carrier proteins were found in salivary glands where the processing of blood and thus release of heme does not occur. Besides, the carrier proteins bind relatively low amounts of heme (in one case only two molecules of heme per protein) compared to their sizes (above 200 kDa).The main aim of this study is the biochemical characterization of a carrier protein from the ornate sheep tick Dermacentor marginatus, hemelipoglycoprotein, with emphasis on its size in native conditions, its glycosylation and identification of its modifying glycans, and examining its carbohydrate-binding specificity.ResultsHemelipoglycoprotein from D. marginatus plasma was purified in native state by immunoprecipitation and denatured using electroelution from SDS-PAGE separated plasma. The protein (290 kDa) contains two subunits with molecular weights 100 and 95 kDa. It is glycosylated by high-mannose and complex N-glycans HexNAc2Hex9, HexNAc2Hex10, HexNAc4Hex7, and HexNAc4Hex8. The purified protein is able to agglutinate red blood cells and has galactose- and mannose-binding specificity. The protein is recognized by antibodies directed against plasma proteins with hemagglutination activity and against fibrinogen-related lectin Dorin M from the tick Ornithodoros moubata.It forms high-molecular weight complexes with putative fibrinogen-related proteins and other unknown proteins under native conditions in tick plasma. Feeding does not increase its amounts in male plasma. The hemelipoglycoprotein was detected also in hemocytes, salivary glands, and gut. In salivary glands, the protein was present in both glycosylated and nonglycosylated forms.ConclusionA 290 kDa hemelipoglycoprotein from the tick Dermacentor marginatus, was characterized. The protein has two subunits with 95 and 100 kDa, and bears high-mannose and complex N-linked glycans. In hemolymph, it is present in complexes with putative fibrinogen-related proteins. This, together with its carbohydrate-binding activity, suggests its possible involvement in tick innate immunity. In fed female salivary glands, it was found also in a form corresponding to the deglycosylated protein.

Evaluation of glycoproteins purified from adult and larval camel ticks (Hyalomma dromedarii) as a candidate vaccine

In order to identify antigens that can help prevent camel tick infestations, three major glycoproteins (GLPs) about 97, 66 and 40 kDa in size were purified from adult and larval Egyptian ticks, Hyalomma (H.) dromedarii, using a single-step purification method with Con-A sepharose. The purified GLPs were evaluated as vaccines against camel tick infestation in rabbits. The rabbits received three intramuscular inoculations of GLPs (20 µg/animal) on days 0, 14, and 28. In the immunoblot analysis, Sera from the immunized rabbits recognized the native GLPs and other proteins from larval and adult H. dromedarii ticks along with those from other tick species such as Rhipicephalus sanguineus but not Ornithodoros moubata. The effects of immunity induced by these GLPs were determined by exposing rabbits to adult H. dromedarii ticks. These results demonstrated that GLP immunization led to a slightly decreased reproductive index and significantly reduced rates of egg hatchability. These results demonstrated that immunization with the purified GLPs can provide protection against infestation by H. dromedarii and some other tick species. Further studies are needed to confirm the effectiveness of immunization with GLPs against other tick species.

Cloning, characterization and diagnostic performance of the salivary lipocalin protein TSGP1 from Ornithodoros moubata

The argasid tick Ornithodoros moubata is distributed throughout South and East Africa and Madagascar, where it colonizes wild and domestic habitats and feeds on warthogs, domestic swine, and humans. This argasid transmits the spirochete Borrelia duttonii, causing East African tick-borne relapsing fever in humans, and the African swine fever virus, which causes a highly lethal haemorrhagic disease in pigs. Tick surveillance and the elimination of O. moubata from synanthropic environments (human dwellings and pigsties) would facilitate the control and prevention of these two diseases. Since direct surveillance methods are impractical in this context, the development of an indirect method for the detection of specific antibodies against tick salivary proteins in samples taken from animal or human hosts living in the area under study would provide a more convenient surveillance and diagnostic tool. Previous work has indicated that the 20A1 salivary antigen of O. moubata could be an optimal candidate for the development of a specific serological test and identified it as an orthologue of the Ornithodoros savignyi TSGP1 lipocalin. The objectives of the present work were to clone, sequence, and molecularly characterize the O. moubata TSGP1, as well as its production as a recombinant protein in order to assess its usefulness as a diagnostic antigen in an ELISA test for tick surveillance. Our results show that O. moubata TSGP1 (OmTSGP1) conserves the tertiary structure of lipocalins and contains the biogenic amine-binding motif. We also show that OmTSGP1 shares 65% sequence identity with the O. savignyi TSGP1, demonstrating that they represent orthologous proteins and suggesting they share identical function as biogenic amine scavengers. A recombinant form of OmTSGP1 was produced, showing 100% sensitivity and 99.4% specificity in an ELISA test for the detection of anti-O. moubata antibodies in pig sera. This recombinant antigen represents a promising epidemiological tool for O. moubata surveillance that may help to implement control measures against O. moubata-borne diseases.

Molecular cloning of a small heat shock protein (sHSPII) from the cattle tick Rhipicephalus ( Boophilus) annulatus salivary gland

Immunoscreening of a cDNA expression library of the Rhipicephalus ( Boophilus) annulatus tick with purified rabbit anti- R annulatus salivary glands antigens polyclonal antibodies led to the identification of a 661 bp sequence. The sequence includes an open reading frame of 543 bp encoding a protein of 180 amino acids with calculated molecular weight of 20.51 kDa, isoelectric point of 9.071 and with no signal sequence. Comparison of the deduced amino acids with protein data bank showed that the identified polypeptide belongs to the alpha crystallin small heat shock proteins superfamily and shows sequence similarity of 62% and 55% to Ixodes scapularis fed tick salivary gland protein and Ornithodoros parkeri alpha-crystallin protein, respectively. Accordingly, this protein was called Ra-sHSPII. The Ra-sHSPII protein was expressed in E. coli under T7 promotor of the pET-30b vector, purified under denaturation conditions and the immunogenicity and cross-reactivity of the recombinant Ra-sHSPII were evaluated. Direct ELISA showed that the Ra-sHSPII is a strong immunogen. In immunoblotting assay the anti-rRa-sHSPII antisera reacted specifically with purified rRa-sHSPII, with several proteins in R. annulatus whole tick, larval and gut protein extracts in addition to Hyalomma dromedarii and Ornithodoros moubata whole tick protein extracts, as examples of hard and soft tick species, respectively. The rRa-sHSPII protein confers thermal protection to other proteins in vitro as found in other sHSPs. E. coli cell extracts containing the protein were protected from heat-denatured precipitation when heated up to 100 °C, whereas extracts from cells not expressing the protein were heat-sensitive at 60 °C.

Cuticular plasticization in the tick,Amblyomma hebraeum(Acari: Ixodidae): possible roles of monoamines and cuticular pH

The degree of plasticization of the alloscutal cuticle of a 'hard' (ixodid) tick, Amblyomma hebraeum, and a 'soft' (argasid) tick, Ornithodoros moubata, was assessed throughout the blood-feeding period. Cuticle viscosity was calculated from rate of creep of cuticle under constant load using a Maxwell model. Feeding-related plasticization (i.e. increased rate of extension under a constant load) occurred in A. hebraeum but not in O. moubata. Maxwell viscosity of unfed A. hebraeum cuticle was relatively high (approximately 720 GPa s) but was significantly lower in feeding ticks. Small partially fed ticks displayed a viscosity of approximately 108 GPa s. Still lower values (42 GPa s) were observed in the largest of the engorged ticks. Following cessation of feeding, there was a significant but limited reversal in viscosity back to approximately 100 GPa s. The water content of cuticle of unfed A. hebraeum (23.4% of wet mass) rose sharply after the onset of feeding and reached a plateau value of 34.0% at a fed/unfed weight ratio of 3 and beyond. Ixodid ticks lay down new endocuticle during the feeding period. The observed increase in cuticle hydration suggests that both old and new cuticles are hydrated during feeding. Monoamines may play an important role in controlling cuticle viscosity. Dopamine (DA) injected into partially fed A. hebraeum caused plasticization. 5-Hydroxytryptamine (serotonin, 5-HT), which induces plasticization in the blood-sucking insect Rhodnius prolixus, had no statistically significant effect on tick cuticle. Octopamine (OA) and tyramine both caused cuticle stiffening (i.e. opposed plasticization). This suggests a possible inhibitory effect but co-injection of OA with DA did not reduce DA-induced plasticization. The mechanism leading to plasticization of tick cuticle may involve a change in cuticular pH. The viscosity of tick cuticle loops was highest at pH 8.0 (389 GPa s) and fell precipitously in the acidic range to a low value of 2.2 GPa s at pH 5.5-5.7. A cuticular pH of approximately 6.5 would account for the lowest viscosity observed under physiological conditions (42.4 GPa s for large, day 0, engorged ticks). The V-ATPase inhibitor, concanamycin A, was a potent inhibitor of DA-induced plasticization. These results are consistent with a model in which DA acts to cause plasticization through transport of H(+) ions into the cuticle. Measurement of cuticular ion (Na(+), K(+), Ca(2+), Mg(2+)) content did not suggest that plasticization is caused by any of these ions. Taken together, our results suggest that the mechanism of cuticular plasticization in feeding A. hebraeum is related to hydration, and involves the transport of H(+) ions into the sub-cuticular space by cells in the hypodermis. Feeding-induced plasticization was not observed in the rapid feeding tick, O. moubata.