Identification of novel tick salivary gland proteins for vaccine development

Methods currently used to control Ixodes scapularis ticks rely principally on acaricidal applications which suffer from a number of limitations. Recently, host vaccination against ticks has been shown to be a promising alternative tick control method. In tick salivary glands, numerous genes are induced during the feeding process. Many of these newly expressed proteins are secreted in tick saliva and may play a role in modulating host immune responses and pathogen transmission. We have performed suppression subtraction hybridization to identify unique I. scapularis salary gland proteins specifically expressed during engorgement. We have cloned and sequenced ten unique salivary gland-associated cDNAs that are up-regulated during feeding. The protein products of these genes represent potential vaccine candidates for use in the control of ticks and to prevent transmission of tick-borne diseases.     

Tick saliva: recent advances and implications for vector competence

Abstract . Secretions of the tick salivary glands are essential to the successful completion of the prolonged feeding of these ectoparasites as well as the conduit by which most tick-borne pathogens are transmitted to the host. In ixodid ticks the salivary glands are the organs of osmoregulation, and excess water from the bloodmeal is returned via saliva into the host. Host blood must continue to flow into the feeding lesion as well as remain fluid in the tick mouthparts and gut. The host's haemostatic mechanisms are thwarted by various anti-platelet aggregatory, anticoagulatory and anti-vasoconstrictory factors in tick saliva. Saliva components suppress the immune and inflammatory response of the host permitting the ticks to remain on the host for an extended period of time and, adventitiously, enhancing the transmission and establishment of tick-borne pathogens. Over the years much work has been done on the numerous enzyme and pharmacological activities found in the tick saliva. The present article reviews the most recent work on salivary gland secretionith special emphasis on how they favour pathogen transmission.     

Identification of SNARE and cell trafficking regulatory proteins in the salivary glands of the lone star tick,Amblyomma americanum (L.)

Prostaglandin E(2) (PGE(2)) stimulates secretion of tick salivary gland proteins via a phosphoinositide signaling pathway and mobilization of intracellular Ca(2+) (). Highly conserved intracellular SNARE (soluble NSF attachment protein receptors) complex proteins are associated with the mechanism of protein secretion in vertebrate and invertebrate neuronal and non-neuronal cells. Proteins in the salivary glands of partially fed female lone star ticks cross-react individually with antibodies to synaptobrevin-2 (vesicle (v)-SNARE), syntaxin-1A, syntaxin-2 and SNAP-25 (target (t)-SNAREs), cytosolic alpha/beta SNAP and NSF (N-ethylmaleimide-sensitive fusion protein), Ca(2+) sensitive synaptotagmin, vesicle associated synaptophysin, and regulatory cell trafficking GTPases Rab3A and nSec1. V-SNARE and t-SNARE proteins form an SDS-resistant, boiling sensitive core complex in the salivary glands. Antibodies to SNARE complex proteins inhibit PGE(2)-stimulated secretion of anticoagulant protein in permeabilized tick salivary glands. We conclude that SNARE and cell trafficking regulatory proteins are present and functioning in the process of PGE(2)-stimulated Ca(2+) regulated protein secretion in tick salivary glands.     

Role of an aquaporin in the sheep tick Ixodes ricinus: Assessment as a potential control target

Ticks undergo tremendous osmoregulatory stress as they take on up to 100 times their body weight in blood, returning about 75% of the ingested water and ions via their saliva into the host. We postulated that water channels, or aquaporins, involved in this mass water transport might be good targets for acaricide development. An aquaporin (IrAQP1) identified in the sheep tick, Ixodes ricinus, was present only in tissues involved in mass water flux, namely the gut, rectal sac and especially abundant in the salivary glands. IrAQP1 was localised by in situ hybridisation in specific cell and acini types, possibly Type III acini, but absent from the type I acini that are responsible for rehydration of ticks in the non-feeding phase. Gene knockdown of IrAQP1 in isolated salivary glands completely inhibited dopamine-stimulated secretion. Further, IrAQP1 knockdown adult females had 50% reduced body weight gains over the first 5 days feeding on an artificial feeding apparatus and 21% at the point of engorgement on hosts. Haemolymph osmolarity was increased in the IrAQP1-knockdown ticks. Importantly, the blood volume ingested per body weight was reduced by 30%. Overall, it would appear that water passage from the gut to the saliva was disrupted and tick guts were simply too “full” to ingest more blood. However, double-stranded RNA interference of IrAQP1 did not affect mortality of the ticks which successfully fed to detachment at day 9. Overall, our data indicate that IrAQP1 plays a pivotal role in blood meal water handling through the gut and salivary gland, and although its disruption by double-stranded RNA interference dramatically affects feeding performance, ticks remained feeding on the host with subsequent potential pathogen transmission and, therefore, IrAQP1 is not a suitable candidate target for tick control.     

Saliva,Salivary Gland,and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) ^1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick ^3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick''s enzootic cycle ^14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva ^9,16-19.As a tick feeds the host typically responds with a strong hemostatic and innate immune response ^11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding ^{3,11,20,21,23}. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens ^3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.     

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

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

Characterization of a novel salivary immunosuppressive protein from Ixodes ricinus ticks.

In tick salivary glands, several genes are induced during the feeding process, leading to the expression of new proteins. These proteins are typically secreted in tick saliva and are potentially involved in the modulation of the host immune and hemostatic responses. In a previous study, the construction and the analysis of a subtractive library led to the identification of Ixodes ricinus immunosuppressor (Iris), a novel protein, differentially expressed in I. ricinus salivary glands during the blood meal. In the present study, the data strongly suggest that this protein is secreted by tick salivary glands into the saliva. In addition, Iris is also found to modulate T lymphocyte and macrophage responsiveness by inducing a Th2 type response and by inhibiting the production of pro-inflammatory cytokines. In conclusion, these results suggest that Iris is an immunosuppressor, which might play an important role in the modulation of host immune response.     

Probing the functional role of tick metalloproteases

Tick saliva assists feeding through a complex mixture of compounds that disarm the host homeostasis processes, such as platelet aggregation, vasoconstriction and blood clotting, as well as innate and acquired immune responses. Although the various properties of tick salivary glands have sparked great interest as candidate sources for anti‐tick vaccines to prevent tick and tick‐borne diseases, antigens that can be useful to induce an immune response against tick bites or the pathogens transmitted by ticks have not yet been developed. Metalloproteases, which have been found in tick saliva, salivary gland, ovary and midgut, play an important role in inflammation, immunomodulation, fibrinolysis, blood protein digestion, nociception, vitellogenesis, remodelling of extracellular matrix and pathogen transmission. A large proportion of tick metalloproteases belong to the metzincin group, whose members characteristically have a highly conserved zinc‐binding motif integrated into the central α helix at the active site, and a methionine‐containing triad called Met‐turn followed by a cysteine‐rich domain at the C‐terminal site. This review discusses specifically the biological aspects of metalloproteases in tick physiology that have been published to date.     

Tick saliva in anti-tick immunity and pathogen transmission

When feeding on vertebrate host ticks (ectoparasitic arthropods and potential vectors of bacterial, rickettsial, protozoal, and viral diseases) induce both innate and specific acquired host-immune reactions as part of anti-tick defenses. In a resistant host immune defense can lead to reduced tick viability, sometimes resulting in tick death. Tick responds to the host immune attack by secreting saliva containing pharmacologically active molecules and modulating host immune response. Tick saliva-effected immunomodulation at the attachment site facilitates both tick feeding and enhances the success of transmission of pathogens from tick into the host. On the other hand, host immunization with antigens from tick saliva can induce anti-tick resistance and is seen to be able to induce immunity against pathogens transmitted by ticks. Many pharmacological properties of saliva described in ticks are shared widely among other blood-feeding arthropods.     

Rhipicephalus appendiculatus feeding on rabbits and cattle: Salivary-gland responses to varying host resistance

AdultRhipicephalus appendiculatus ticks were fed as three sequential infestations on both rabbits and cattle. The feedings at first infestation on naive hosts were optimum for the ticks, whereas at third infestation the hosts were resistant, as expressed by reduced tick feeding performance. Ticks from naive and resistant hosts were examined for histological differences of salivary glands. In ticks fed on resistant rabbits there was a large increase in the synthesis of glycoprotein secretory granules in thec ₁ cells compared with ticks fed on naive rabbits. In ticks fed on naive and resistant cattle, the activity of thec ₁ cells was less than in ticks fed on naive and resistant rabbits. It was concluded that the salivary glands are able to respond selectively to conditions at the feeding site, and that this may be advantageous to the tick.     

Tick antigens recognized by serum from a guinea pig resistant to infestation with the tick Rhipicephalus appendiculatus

Immune resistance to infestation by an ixodid tick, Rhipicephalus appendiculatus, the vector of the cattle disease East Coast Fever, was induced in a guinea pig by repeated tick infestation. This resistance is expressed as the ability of the host to interfere with tick feeding. Resistance to ixodid tick feeding is an acquired response mediated by host antibody. We report the use of antibodies from a resistant host animal, in immunoblotting, to characterize the tick antigens recognized. The major tick antigens identified had molecular weights of 120,000, 94,000, 88,000, 77,000, 58,000, 46,000, 35,000, 31,000, 28,000, 25,000, 20,000 and 16,000. Most of these antigens were found in tick salivary glands. The presence and concentration of many tick salivary antigens appeared to vary with relation to the tick feeding cycle. Many of the antigens present in salivary glands were also detected in tick cement. Tick gut extract, although a poorer source of antigens, contained more of the 31,000 dalton antigen than salivary glands. Larval and nymphal tick extract lacked many of the antigens present in adult ticks. The data suggest that tick resistance is a complex phenomenon probably elicited by several different tick antigens.     

Tick modulation of host immunity: an important factor in pathogen transmission.

Immunological interactions at the tick host interface involve innate and specific acquired host immune defenses and immunomodulatory countermeasures by the tick. Tick feeding stimulates host immune response pathways involving antigen-presenting cells, cytokines, B-cells, T-cells, circulating and homocytotropic antibodies, granulocytes, and an array of biologically active molecules. In response to host immune defenses, tick-mediated host immunosuppressive countermeasures inhibit: host antibody responses; complement activation; T-cell proliferation; and cytokine elaboration by macrophages and Th1-lymphocytes. Immunosuppressive proteins identified in tick salivary glands and saliva have been partially characterised. Tick-induced host immunosuppression facilitates blood meal acquisition and is an important factor in the transmission/establishment of the tick-borne disease-causing agent, Borrelia burgdorferi. A novel strategy for control of tick-borne pathogens is proposed.     

The impact of gene knock-down and vaccination against salivary metalloproteases on blood feeding and egg laying by Ixodes ricinus

Two cDNAs coding homologous putative metalloproteases (Metis 1 and Metis 2, expected molecular weights of 55.6 and 56.0kDa, respectively) were identified from the hard tick Ixodes ricinus. The expression of Metis genes was induced in salivary glands during tick blood meal. RNA interference was used to assess the role of both Metis 1 and Metis 2 in tick feeding. It was found that salivary gland extracts lacking Metis 1-2 had a restricted ability to interfere with fibrinolysis. RNAi against Metis 1-2 also induced a high mortality rate. An immune reaction was raised in repeatedly bitten animals against Metis 1 and 2. Vaccination of hosts with the recombinant Metis 1 protein produced in a eukaryotic system partially interfered with completion of the blood meal. Although vaccination did not alter the survival rate or feeding time of ticks, their weight gain and oviposition rate were reduced. This will affect their reproductive fitness in the field. We believe this is the first report of an anti-tick vaccine trial using a metalloprotease derived from I. ricinus.