Ixodes ricinus immunogenic saliva protein, homologue to Amblyomma americanum AV422: Determining its potential for use in tick bite confirmation.

Tick bites often go unnoticed, so specific reliable tests are needed to confirm them for prompt diagnosis and treatment of tick-borne diseases. One of the promising candidates for developing such a test is AV422, a tick saliva protein that has been conserved across tick genera. In this study, we demonstrate the potential of the AV422 homologue from Ixodes ricinus to be used for tick bite detection for both Prostriata and Metastriata. We expressed recombinant (r) I. ricinus (Ir) AV422 in E. coli and subjected it to Western blot analysis using rat antibodies to saliva proteins of both I. ricinus (Prostriata) and Dermacentor reticulatus (Metastriata) larvae. Our data demonstrate that rIrAV422 specifically bound to antibodies from sera of rats used for both I. ricinus and D. reticulatus larvae feeding, but not to antibodies from control serum, emphasizing its specificity since tick bites were the sole cause of sera reactivity.

Defending the fort: a role for defensin-2 in limiting Rickettsia montanensis infection of Dermacentor variabilis.

The importance of tick defensins is evidenced by their expression in a wide variety of tick tissues and prevalence across many tick genera. To date, the functional and biological significance of defensin-2 as a rickettsiastatic or rickettsiacidal antimicrobial peptide has not been addressed. In a previous study, defensin-2 transcription was shown to increase in Dermacentor variabilis ticks challenged with Rickettsia montanensis. In the present study, the hypothesis that defensin-2 is functional as a rickettsiastatic and/or rickettsiacidal antimicrobial peptide is tested. We show that defensin-2 plays a role in reducing burden after acquisition of Rickettsia montanensis through capillary feeding. Moreover, defensin-2 is shown to associate with R. montanensis in vitro and in vivo, causing cytoplasmic leakiness.

Gene expression of tissue-specific molecules in ex vivo Dermacentor variabilis (Acari: Ixodidae) during rickettsial exposure.

Ticks serve as both vectors and the reservoir hosts capable of transmitting spotted fever group Rickettsia by horizontal and vertical transmission. Persistent maintenance of Rickettsia species in tick populations is dependent on the specificity of the tick and Rickettsia relationship that limits vertical transmission of particular Rickettsia species, suggesting host-derived mechanisms of control. Tick-derived molecules are differentially expressed in a tissue-specific manner in response to rickettsial infection; however, little is known about tick response to specific rickettsial species. To test the hypothesis that tissue-specific tick-derived molecules are uniquely responsive to rickettsial infection, a bioassay to characterize the tick tissue-specific response to different rickettsial species was used. Whole organs of Dermacentor variabilis (Say) were exposed to either Rickettsia montanensis or Rickettsia amblyommii, two Rickettsia species common, or absent, in field-collected D. variabilis, respectively, for 1 and 12 h and harvested for quantitative real time-polymerase chain reaction assays of putative immune-like tick-derived factors. The results indicated that tick genes are differently expressed in a temporal and tissue-specific manner. Genes encoding glutathione S-transferase 1 (dvgst1) and Kunitz protease inhibitor (dvkpi) were highly expressed in midgut, and rickettsial exposure downregulated the expression of both genes. Two other genes encoding glutathione S-transferase 2 (dvgst2) and beta-thymosin (dvpbeta-thy) were highly expressed in ovary, with dvbeta-thy expression significantly downregulated in ovaries exposed to R. montanensis, but not R. amblyommii, at 12-h postexposure, suggesting a selective response. Deciphering the tissue-specific molecular interactions between tick and Rickettsia will enhance our understanding of the key mechanisms that mediate rickettsial infection in ticks.

Prostaglandin E(2) in tick saliva regulates macrophage cell migration and cytokine profile.

BACKGROUND: Ticks are obligate hematophagous ectoparasites that suppress the host's immune and inflammatory responses by secreting immuno-modulatory and anti-inflammatory molecules in their saliva. In previous studies we have shown that tick salivary gland extract (SGE) and saliva from Dermacentor variabilis have distinct effects on platelet-derived growth factor (PDGF)-stimulated IC-21 macrophage and NIH3T3-L1 fibroblast migration. Since tick saliva contains a high concentration of prostaglandin E2 (PGE2), a potent modulator of inflammation, we used a PGE2 receptor antagonist to evaluate the role of PGE2 in the different migratory responses induced by saliva and its impact on macrophage cytokine profile. METHODS: Adult ticks were fed on female New Zealand white rabbits for 5-8 days. Female ticks were stimulated with dopamine/theophylline to induce salivation and saliva was pooled. Competitive enzyme immunoassays (EIA) were used to measure saliva PGE2 content and the changes in macrophage intracellular cyclic adenosine monophosphate (cAMP) levels. The effects of tick saliva on macrophage and fibroblast migration were assessed in the absence and presence of the PGE2 receptor antagonist, AH 6809, using blind well chamber assays. A cytokine antibody array was used to examine the effects of tick saliva on macrophage cytokine secretion. Statistical significance was determined by one-way ANOVA; Student Newman-Kuels post-test was used for multiple comparisons. RESULTS: The saliva-induced increase in PDGF-stimulated macrophage migration was reversed by AH 6809. The inhibition of PDGF-stimulated fibroblast migration by saliva was also antagonist-sensitive. Tick saliva induced macrophages to secrete copious amounts of PGE2, and conditioned medium from these cells caused an AH 6809-sensitive inhibition of stimulated fibroblast migration, showing that macrophages can regulate fibroblast activity. We show that tick saliva decreased the secretion of the pro-inflammatory cytokines regulated and normal T cell expressed and secreted (RANTES/CCL5), tumor necrosis factor-alpha (TNF-α), and soluble TNF receptor I (sTNFRI) through a PGE2-dependent mechanism mediated by cAMP. Saliva had similar effects on lipopolysaccharide (LPS) stimulated macrophages. CONCLUSIONS: Our data show that ticks utilize salivary PGE2 to subvert the ability of macrophages to secrete pro-inflammatory mediators and recruit fibroblasts to the feeding lesion, therefore inhibiting wound healing.

Immunoproteomic identification of antigenic salivary biomarkers detected by Ixodes ricinus-exposed rabbit sera.

Ixodes ricinus, the primary vector of tick-borne disease in Europe, is currently expanding its distribution area and its activity in many countries. Antibody responses to tick salivary antigens have been proposed as an alternative marker of exposure to tick bites. However, the identification of the I. ricinus corresponding antigens remains elusive. Using rabbits artificially exposed to I. ricinus and 2 other European tick species (Rhipicephalus sanguineus and Dermacentor reticulatus) as controls, a cross-comparison of IgG profiles was performed against protein salivary gland extracts (pSGE) from these 3 tick species using immunoblots. Immunoblot analysis highlighted a singularity in the immune patterns according to tick species exposure and pSGE antigen source. Two protein bands were detected against I. ricinus pSGE only in rabbits exposed to I. ricinus bites. An immunoproteomic approach based on a fluorescence detection method was developed to unambiguously identify corresponding antigenic spots on 2-D gels. Among the unique I. ricinus salivary antigenic proteins detected by sera from rabbits exposed to this tick species, I. ricinus calreticulin was identified. Although tick calreticulin was previously proposed as a potential antigenic marker following exposure to ticks (particularly in North American tick species), the present study suggested that Ixodes calreticulin does not appear to be cross-recognized by the 2 other tick genera tested. Additional experiments are needed to confirm the use of I. ricinus calreticulin salivary protein as a potential discriminant antigenic biomarker to Ixodes tick exposure.

Identification of salivary antigenic markers discriminating host exposition between two European ticks: Rhipicephalus sanguineus and Dermacentor reticulatus.

To succeed blood meal, ticks inject salivary proteins to mammalian hosts, eliciting an antibody response against these foreign antigens. Although this immune response has been proposed as a surrogate marker of exposure to tick bites, identification of the corresponding antigens remains elusive. For this aim, a comparison by immunoblots of the kinetic IgG responses to protein salivary gland extracts from two European tick species, Rhipicephalus sanguineus or Dermacentor reticulatus, in rabbits was performed. A singularity in the immune patterns was observed according to rabbit exposure status and depending on the antigen source. Six and five bands were found specifically associated to R. sanguineus and to D. reticulatus exposures, respectively. The identity of these salivary antigenic proteins was determined using an original immunoproteomic approach. The utilization of these tick salivary proteins as biomarker candidates to discriminate R. sanguineus and/or D. reticulatus tick exposure or to develop anti-tick vaccines is discussed.

Pyrosequencing and characterization of immune response genes from the American dog tick, Dermacentor variabilis (L.).

Ticks continue to be a threat to animal and human health, and new and novel control strategies are needed for ticks and tick-borne pathogens. The characterization of the tick-pathogen interface and the tick immune response to microbial infections is fundamental toward the formulation of new control strategies for ticks and the pathogens they transmit. Our overall hypothesis for this research is that the tick immune system manages the maintenance of pathogens. Therefore, discovery of tick immune response genes may provide targets for novel control strategies directed toward reducing vector competency and pathogen transmission. In these studies, 454 pyrosequencing, a high-throughput genomic sequencing method was used to discover tick genes expressed in response to bacterial and fungal infections. Expressed sequence tags (ESTs) were analysed from Dermacentor variabilis ticks that had been injected with bacteria (Escherichia coli, Bacillus subtilis, Micrococcus luteus) or fungi (Saccharomyces cerevisiae and Candida albicans) and ticks that were naturally infected with the intracellular bacterium, Anaplasma marginale. By this approach, ESTs were assembled into 5995 contigs. Contigs fell into the five main functional categories of metabolism, genetic information processing, environmental information processing, cellular processes and human diseases. We identified more than 30 genes that are likely to encode for proteins involved in tick immune function. We further analysed by reverse transcriptase PCR (RT-PCR) the expression of 22 of these genes in each of our bacterial or fungal treatment groups and found that seven were up-regulated. Up-regulation of these seven genes was confirmed for bacterial, but not fungal treatment by quantitative PCR (qPCR). One of these products was novel, encoding a new tick defensin. Our results clearly demonstrate the complexities of the tick immune system and mark new directions for further study and characterization of proteins that modulate microbial infections in the American dog tick.

Subolesin expression in response to pathogen infection in ticks.

BACKGROUND: Ticks (Acari: Ixodidae) are vectors of pathogens worldwide that cause diseases in humans and animals. Ticks and pathogens have co-evolved molecular mechanisms that contribute to their mutual development and survival. Subolesin was discovered as a tick protective antigen and was subsequently shown to be similar in structure and function to akirins, an evolutionarily conserved group of proteins in insects and vertebrates that controls NF-kB-dependent and independent expression of innate immune response genes. The objective of this study was to investigate subolesin expression in several tick species infected with a variety of pathogens and to determine the effect of subolesin gene knockdown on pathogen infection. In the first experiment, subolesin expression was characterized in ticks experimentally infected with the cattle pathogen, Anaplasma marginale. Subolesin expression was then characterized in questing or feeding adult ticks confirmed to be infected with Anaplasma, Ehrlichia, Rickettsia, Babesia or Theileria spp. Finally, the effect of subolesin knockdown by RNA interference (RNAi) on tick infection was analyzed in Dermacentor variabilis males exposed to various pathogens by capillary feeding (CF). RESULTS: Subolesin expression increased with pathogen infection in the salivary glands but not in the guts of tick vector species infected with A. marginale. When analyzed in whole ticks, subolesin expression varied between tick species and in response to different pathogens. As reported previously, subolesin knockdown in D. variabilis infected with A. marginale and other tick-borne pathogens resulted in lower infection levels, while infection with Francisella tularensis increased in ticks after RNAi. When non-tick-borne pathogens were fed to ticks by CF, subolesin RNAi did not affect or resulted in lower infection levels in ticks. However, subolesin expression was upregulated in D. variabilis exposed to Escherichia coli, suggesting that although this pathogen may induce subolesin expression in ticks, silencing of this molecule reduced bacterial multiplication by a presently unknown mechanism. CONCLUSIONS: Subolesin expression in infected ticks suggested that subolesin may be functionally important for tick innate immunity to pathogens, as has been reported for the akirins. However, subolesin expression and consequently subolesin-mediated innate immunity varied with the pathogen and tick tissue. Subolesin may plays a role in tick innate immunity in the salivary glands by limiting pathogen infection levels, but activates innate immunity only for some pathogen in the guts and other tissues. In addition, these results provided additional support for the role of subolesin in other molecular pathways including those required for tissue development and function and for pathogen infection and multiplication in ticks. Consequently, RNAi experiments demonstrated that subolesin knockdown in ticks may affect pathogen infection directly by reducing tick innate immunity that results in higher infection levels and indirectly by affecting tissue structure and function and the expression of genes that interfere with pathogen infection and multiplication. The impact of the direct or indirect effects of subolesin knockdown on pathogen infection may depend on several factors including specific tick-pathogen molecular interactions, pathogen life cycle in the tick and unknown mechanisms affected by subolesin function in the control of global gene expression in ticks.

Inducing active and passive immunity in sheep to paralysis caused by Dermacentor andersoni.

Arcott sheep were evaluated as a model for studying active and passive immunity to tick paralysis caused by Dermacentor andersoni (Stiles). The incidence of tick paralysis in sheep increased from 0 at doses < or = 0.33 ticks per kg to 100% at > or = 0.8 ticks per kg. The dose required for 50% paralysis was 0.42 ticks per kg. Expressing dose as a ratio of initial ticks per unit body weight removed differences in response due to sheep weight. The interval from infestation to paralysis decreased from >12 d at 0.4 ticks per kg to <8 d at 1.3 ticks per kg. After exposure to a paralyzing doses of ticks, the incidence of paralysis varied among sheep that were naive (six of six, 100% paralysis), previously paralyzed (zero of six, 0% paralysis), and passively immunized with an intravenous treatment of 300 ml of serum from immune cattle (two of six, 33% paralysis). Sheep that were actively immunized by previous exposure had antibodies to a greater number of tick salivary antigens compared with those that were not immune. Antibodies to a 43.3-kDa antigen had 72% agreement with immunity to paralysis, and a sensitivity and specificity of 0.60 and 0.88, respectively. In conclusion, previously paralyzed sheep had developed antibodies against D. andersoni and were not susceptible to subsequent paralysis, whereas passive immunization conferred protection against paralysis in only some sheep.

Cattle can develop immunity to paralysis caused by Dermacentor andersoni.

Cattle exposed to a paralyzing strain of Dermacentor andersoni (Stiles) were all paralyzed during an initial exposure, but the incidence of paralysis decreased to 17 and 0% after two subsequent exposures to virulent flat ticks. Cattle with a single exposure to paralyzing ticks became paralyzed when challenged with ticks that had been prefed on cattle. Western blots indicated that cattle developed antibody responses to 13 antigens in paralyzing tick saliva. The likelihood of paralysis was inversely related to the number of saliva proteins that cattle developed antibody responses to. Cattle challenged with prefed ticks developed antibody response to fewer saliva antigens than cattle challenged with flat ticks. Variation in tick dose did not influence the expression of paralysis. Daily survival of ticks was similar on all groups of cattle, and tick weight was not reduced on previously challenged cattle, indicating immunity developed to the paralysis toxin rather than tick feeding. Four saliva antigens (molecular weights ranging from 36.9 to 42.2 kDa) were associated with the development of immunity to paralysis.

Evaluation of sequential coinfection with Anaplasma phagocytophilum and Anaplasma marginale in cattle.

OBJECTIVE: To determine whether sequelae of infection differed among single versus double infection with Anaplasma phagocytophilum or Anaplasma marginale, with and without tick salivary extract, in cattle. ANIMALS: Eighteen 13-month old steers. PROCEDURES: Treatment groups of 3 cattle each included A marginale inoculated ID followed on day 35 by A phagocytophilum without tick saliva, A phagocytophilum followed on day 10 by A marginale without tick saliva, A marginale followed on day 35 by A phagocytophilum with tick saliva, A phagocytophilum followed on day 10 by A marginale with tick saliva, tissue culture control injection, and tick saliva control injection. Infection was monitored via clinical observations, CBC, serologic testing, and PCR analysis of blood and tissues. RESULTS: Infected cattle had significantly reduced weight gain. Anemia occurred 25 to 32 days after A marginale infection, which was attenuated by tick saliva. Parasitism was greater if cattle had not previously been inoculated with A phagocytophilum. Nine of the 12 treated cattle had positive results of PCR analysis for A phagocytophilum from at least 1 blood sample. Five tissue samples had positive results of PCR analysis for A phagocytophilum; PCR results for A marginale were positive in spleen, lung, lymph node, heart, and ear skin of infected cattle. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated an important biological interaction between A marginale and A phagocytophilum infection as well as with tick saliva in disease kinetics and severity in cattle, which may be important for interpretation of diagnostic tests and management of disease in areas where both pathogens occur.

Silencing expression of the defensin, varisin, in male Dermacentor variabilis by RNA interference results in reduced Anaplasma marginale infections.

Antimicrobial peptides, including defensins, are components of the innate immune system in ticks that have been shown to provide protection against both gram-negative and gram-positive bacteria. Varisin, one of the defensins identified in Dermacentor variabilis, was shown to be produced primarily in hemocytes but transcript levels were also expressed in midguts and other tick cells. In this research, we studied the role of varisin in the immunity of ticks to the gram-negative cattle pathogen, Anaplasma marginale. Expression of the varisin gene was silenced by RNA interference (RNAi) in which male ticks were injected with varisin dsRNA and then allowed to feed and acquire A. marginale infection on an experimentally-infected calf. Silencing expression of varisin in hemocytes, midguts and salivary glands was confirmed by real time RT-PCR. We expected that silencing of varisin would increase A. marginale infections in ticks, but the results demonstrated that bacterial numbers, as determined by an A. marginale msp4 quantitative PCR, were significantly reduced in the varisin-silenced ticks. Furthermore, colonies of A. marginale in ticks used for RNAi were morphologically abnormal from those seen in elution buffer injected control ticks. The colony shape was irregular and in some cases the A. marginale appeared to be free in the cytoplasm of midgut cells. Some ticks were found to be systemically infected with a microbe that may have been related to the silencing of varisin. This appears to be the first report of the silencing of expression of a defensin in ticks by RNAi that resulted in reduced A. marginale infections.

Using RNA interference to determine the role of varisin in the innate immune system of the hard tick Dermacentor variabilis (Acari: Ixodidae).

Defensins are an important component of the innate immune system of ticks. These small peptides are produced by various genera of ticks, and expressed in various tissues. In this study we used RNA interference to silence the expression of the defensin varisin produced by the hemocytes of the American dog tick, Dermacentor variabilis. Ticks were injected with double stranded varisin RNA prior to being placed on a rabbit. After feeding, the ticks were removed, bled, and the hemolymph plasma and hemocytes separated. Hemocytes were screened for the presence (or absence) of both varisin transcript and peptide. Varisin peptide was below detectable levels and the transcript showed a greater than 99% knockdown. The antimicrobial activity of the hemolymph plasma was reduced 2-4 fold compared to that of control injected ticks indicating varisin accounts for a large portion of the antimicrobial activity of the hemolymph.

Immunomodulatory arsenal of nymphal ticks.

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

Rickettsia peacockii, an endosymbiont of Dermacentor andersoni, does not elicit or inhibit humoral immune responses from immunocompetent D. andersoni or Ixodes scapularis cell lines.

Ixodes scapularis and Dermacentor andersoni cell lines were stimulated with heat-killed Escherichia coli and Micrococcus luteus to investigate whether infection by Rickettsia peacockii, an endosymbiont of D. andersoni, modifies humoral immune responses. Radial diffusion assays, western blotting, flow cytometry, and quantitative reverse-transcription PCR were used to determine if expression of bacteriolytic peptides, including lysozyme and defensin, was upregulated by bacterial stimulation or infection with R. peacockii. The I. scapularis line IDE12 upregulated expression of lysozyme and defensin following stimulation. The D. andersoni cell line DAE15 also expressed defensin and lysozyme, but only lysozyme was upregulated by bacterial stimulation. R. peacockii infection alone, or in cells stimulated with bacteria, did not modify defensin or lysozyme expression in either cell line. These results suggest tick endosymbionts may avoid recognition by the tick immune system, and infection may not affect humoral immune responses to bacteria not normally associated with ticks.

New tick defensin isoform and antimicrobial gene expression in response to Rickettsia montanensis challenge.

Recent studies aimed at elucidating the rickettsia-tick interaction have discovered that the spotted fever group rickettsia Rickettsia montanensis, a relative of R. rickettsii, the etiologic agent of Rocky Mountain spotted fever, induces differential gene expression patterns in the ovaries of the hard tick Dermacentor variabilis. Here we describe a new defensin isoform, defensin-2, and the expression patterns of genes for three antimicrobials, defensin-1 (vsnA1), defensin-2, and lysozyme, in the midguts and fat bodies of D. variabilis ticks that were challenged with R. montanensis. Bioinformatic and phylogenetic analyses of the primary structure of defensin-2 support its role as an antimicrobial. The tissue distributions of the three antimicrobials, especially the two D. variabilis defensin isoforms, are markedly different, illustrating the immunocompetence of the many tissues that R. montanensis presumably invades once acquired by the tick. Antimicrobial gene expression patterns in R. montanensis-challenged ticks suggest that antimicrobial genes play a role during the acquisition-invasion stages in the tick.

Characterization of Dermacentor variabilis molecules associated with Rickettsial infection.

To ultimately define the virulence factors of rickettsiae, an understanding of the biology of the organism is essential. Comprehension of the pathogen-human interaction is critical to the development of control measures; and, in the case of vector-borne diseases, the role of the vector in maintaining and transmitting pathogens to vertebrate hosts is crucial to ultimate control. Recent studies have identified tick molecules that are likely involved in the tick-rickettsiae interchange, including tick response to infection and possible molecules exploited by rickettsiae during transmission events. We have further characterized several tick-derived molecules, including a histamine release factor, serine proteases, and lysozymes.

Capillary tube feeding system for studying tick-pathogen interactions of Dermacentor variabilis (Acari: Ixodidae) and Anaplasma marginale (Rickettsiales: Anaplasmataceae).

A capillary tube feeding (CTF) system was adapted for studying the interaction between Dermacentor variabilis (Say) and the rickettsial cattle pathogen Anaplasma marginale Theiler. A. marginale undergoes a complex developmental cycle in ticks that begins in midguts and ends by transmission from salivary glands. In this CTF system, male D. variabilis were fed A. marginale-infected blood or cultured tick cells. Ticks that fed on highly rickettsemic calves developed midgut and salivary gland infections as detected by PCR, whereas ticks that were fed from capillary tubes on the same blood developed only midgut infections. An unexpected result of capillary tube feeding was that antibodies against the A. marginale adhesin, major surface protein la, enhanced midgut infections and caused cell culture-derived A. marginale to infect midguts. Another unexpected result was the infection of the midguts of the nonvector tick Amblyomma americanum (L.), after capillary tube feeding on infected bovine blood. The gut cell response of ticks to A. marginale, as determined from SDS-polyacrylamide gel electrophoresis protein profiles, did not differ when ticks were fed infected or uninfected cells from capillary tubes. Selected protein bands, as identified by tryptic digestion-mass spectrometry, contained mostly proteins of bovine origin, including bovine albumin, undigested alpha- and beta-chain hemoglobin and hemoglobin fragments. Although infection of ticks by A. marginale CTF system was not the same as infection by feeding on cattle, the results obtained demonstrated the potential use of this system for identifying aspects of pathogen-vector interactions that are not readily recognized in naturally feeding ticks.

Tick modulation of the in-vitro expression of adhesion molecules by skin-derived endothelial cells.

As a tick feeds, its saliva induces innate and acquired immune responses in the host, including leucocyte infiltration into the bite site. Tick salivary glands produce molecules, however, that counteract many host defences against blood feeding. The effects of salivary-gland extracts (SGE) of Dermacentor andersoni and Ixodes scapularis on the expression of various adhesion molecules [E-selectin, P-selectin, intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1)] by the sEND.1 cell line (which is based on cells from the subcutaneous tissue of mice) have now been investigated in vitro. The effects were found to differ with the tick species. The SGE of D. andersoni significantly down-regulated the expression of ICAM-1, whereas a similar extract prepared from I. scapularis significantly reduced the expression of P-selectin and VCAM-1. Tick salivary proteins therefore appear to have direct effects on adhesion-molecule expression, in addition to their previously established roles in down-regulating the pro-inflammatory cytokines that activate endothelial cells. It remains unclear exactly how the reduction of adhesion-molecule expression in the host's endothelial cells benefits the feeding tick but it may alter leucocyte migration to the bite site and/or reduce antigen presentation by the endothelial cells. It may also modulate the interactions between the host's leucocytes and any tick-borne pathogens, during initial infection of the endothelium.

Sugar epitopes as potential universal disease transmission blocking targets.

One promising method to prevent vector-borne diseases is through the use of transmission blocking vaccines (TBVs). However, developing several anti-pathogen TBVs may be impractical. In this study, we have identified a conserved candidate carbohydrate target in the midguts of several Arthropod vectors. A screen of the novel GlycoChip glycan array found that the anti-carbohydrate malaria transmission blocking monoclonal antibody (MG96) preferentially recognized D-mannose (alpha) and the type II lactosamine disaccharide. The specificity for D-mannose was confirmed by competition ELISA using alpha-methyl mannoside as inhibitor. Con A, which identifies terminal mannose residues, did not inhibit MG96 reactivity with mosquito midgut lysates, suggesting that Con A has differential recognition of this monosaccharide. However, the jack bean lectin, Jacalin, which recognizes D-mannose (alpha), d-galactose (alpha/beta) and the T antigen, not only displays a similar banding profile to that recognized by MG96 on immunoblot but was also shown to effectively inhibit MG96. Wheat-germ agglutinin, which recognizes N-acetyllactosamine units, only partially inhibited MG96 reactivity. This highlights the contribution of both glycan moieties to the MG96 epitope or glycotope. Enzyme deglycosylation results suggest that MG96 recognizes a mannose alpha1-6 substitution on an O-linked oligosaccharide. Taken together, the data suggest that MG96 recognizes a discontinuous glycotope composed of Manalpha1-6 proximal to Galbeta1-4GlcNAc-alpha-O-R glycans on arthropod vector midguts. As such, these glycotopes may represent potential transmission blocking vaccine targets for a wide range of vector-borne pathogens.