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.     

Saliva-activated transmission (SAT) of Thogoto virus: dynamics of SAT factor activity in the salivary glands ofRhipicephalus appendiculatus,Amblyomma variegatum,andBoophilus microplus ticks

Thogoto (THO) virus is transmitted from infected to uninfected ticks when co-feeding on uninfected guinea-pigs, even though the guinea-pigs do not develop a detectable viraemia. This form of non-viraemic transmission is potentiated by a factor (s) secreted by the saliva of ticks and hence has been termed saliva-activated transmission (SAT). The synthesis of the SAT factor by the salivary glands of three ixodid tick species was determined by placing uninfected nymphal ticks on guineapigs that were subsequently inoculated with a mixture of THO virus and salivary gland extract (SGE) derived from one of the tick species. SAT factor activity was measured by determining the number of nymphs that acquired THO virus. For the three-host ixodid species,Rhipicephalus appendiculatus andAmblyomma variegatum, maximum enhancement of THO virus transmission was observed when salivary glands were derived from uninfected, female ticks that had fed for a period of 6 or 8 days, respectively. In contrast, when salivary glands were derived form uninfected femaleBoophilus microplus, a one-host ixodid tick species, enhancement of THO virus transmission was observed throughout the tick feeding period. Thus, the natural feeding behaviour of ticks appears to be an important factor in determining the relative importance of these vectors in mediating SAT.     

Changes in dopamine-sensitive adenylate cyclase activity in salivary glands of female lone star ticks,Amblyomma americanum (L.), during feeding

The activity of dopamine-sensitive adenylate cyclase in feeding female ixodid tick salivary glands was dependent on the state of tick feeding. Activity was significantly greater than the “basal” activity in salivary glands from ticks at all stages of tick feeding. Enzyme activity was not detected in the glands of unfed females. Enzyme activity reached a peak in glands of ticks weighing approx. 200 mg then declined as ticks increased in weight beyond 200 mg to repletion. Replete ticks (detached from the host for 12–24 hr) had similar levels of basal and dopamine-stimulated adenylate cyclase activity as that measured in salivary glands of high weight (>200 mg) ticks. Enzyme activity was 19–62% less in glands from ticks feeding on hosts that had been parasitized 2–4 months earlier by lone star ticks.     

Acquired resistance to ixodid ticks induced by tick cement antigen

Antisera from guinea pigs made resistant to infestation with an ixodid tick of east and central Africa,Rhipicephalus appendiculatus, were used to identify the tick antigens they recognized by immunoblotting. Most of the antigens were found in tick salivary glands and in tick attachment cement. Antisera fromR. appendiculatus-resistant guinea pigs also recognized some salivarygland antigens in ticks of other species (R. pulchellus, R. evertsi, Amblyomma variegatum andA. gemma). Antibodies against the most strongly recognizedR. appendiculatus antigen, a 20-kDa molecule, were only poorly reactive with similar-sized molecules in the other ticks. A 94-kDa antigen, which appeared to have broader cross-reactivity, was purified fromR. appendiculatus attachment cement, and a monospecific rabbit serum was raised against it. This antiserum clearly recognized a molecule of similar molecular weight inR. pulchellus andR. evertsi. Intravenous inoculation of rabbits with the purified molecule elicited delayed-type hypersensitivity to the antigen. The hypersensitive rabbits demonstrated resistance to feeding ofR. appendiculatus ticks but slight enhanced feeding ofR. pulchellus ticks. These results are discussed with respect to their relevance for artificial induction of tick-feeding resistance.     

Dot-ELISA assessment of guinea pig antibody responses to repeated Dermacentor andersoni infestations

Acquired resistance to ixodid tick infestation is expressed by cattle and laboratory animals. Humoral factors appear to be involved in host acquired resistance to tick bite; however, specific immune responses have yet to be fully characterized. This study examined tick resistance expressed by Hartley guinea pigs upon repeated infestation with Dermacentor andersoni, and describes longitudinal development of antigen specific immunoglobulin over approximately 180 days. Guinea pigs were infested either 4 times with D. andersoni adults, or twice with nymphs. Both infestation groups, adults and nymphs, demonstrated a significant level of resistance to re-infestation, following initial exposure. Dot enzyme-linked immunosorbent assay (Dot-ELISA) was employed to detect antibody reactive with D. andersoni salivary gland antigens (SGA). Animals infested with adults had antibody that increased at a relatively constant rate until the fourth infestation, which was differentiated by a sharp increase in titer, that was maintained for approximately 2 wk. Guinea pigs that received nymph infestations had SGA-specific antibody; however, titers were lower than those in the adult infestation group. Antibody levels continued to increase approximately 80 days beyond the final (second) infestation for this group. A direct correlation between resistance and antibody titer was not evident, since resistance was relatively stable after the second infestation in both infestation groups, and tick-specific immunoglobulin levels continually increased.     

Functional analysis of the Borrelia burgdorferi bba64 gene product in murine infection via tick infestation

Borrelia burgdorferi, the causative agent of Lyme borreliosis, is transmitted to humans from the bite of Ixodes spp. ticks. During the borrelial tick-to-mammal life cycle, B. burgdorferi must adapt to many environmental changes by regulating several genes, including bba64. Our laboratory recently demonstrated that the bba64 gene product is necessary for mouse infectivity when B. burgdorferi is transmitted by an infected tick bite, but not via needle inoculation. In this study we investigated the phenotypic properties of a bba64 mutant strain, including 1) replication during tick engorgement, 2) migration into the nymphal salivary glands, 3) host transmission, and 4) susceptibility to the MyD88-dependent innate immune response. Results revealed that the bba64 mutant's attenuated infectivity by tick bite was not due to a growth defect inside an actively feeding nymphal tick, or failure to invade the salivary glands. These findings suggested there was either a lack of spirochete transmission to the host dermis or increased susceptibility to the host's innate immune response. Further experiments showed the bba64 mutant was not culturable from mouse skin taken at the nymphal bite site and was unable to establish infection in MyD88-deficient mice via tick infestation. Collectively, the results of this study indicate that BBA64 functions at the salivary gland-to-host delivery interface of vector transmission and is not involved in resistance to MyD88-mediated innate immunity.     

Amblyomma americanum: identification of tick salivary gland antigens from unfed and early feeding females with comparisons to Ixodes dammini and Dermacentor variabilis

Salivary gland antigens involved in host resistance to tick feeding by Amblyomma americanum (lone star tick) have been identified. Gland extracts from unfed and partially fed 12-, 48-, 72-, 96-, and 120-hr females and their corresponding midgut tissues were analyzed by immunoblotting with sera from naturally immune and hyperimmune sheep and rabbits. Polypeptides at 90, 75, 58, 45, 33, and 23 kDa from the salivary glands of A. americanum females were consistently observed with antibodies from both sheep and rabbits. No antigens unique to tick midgut tissue were detected with immune sera. Female Dermacentor variabilis and Ixodes dammini shared 90- and 45-kDa salivary gland antigens with A. americanum, and these may represent conserved polypeptides. We speculate that some of the salivary gland antigens represent components of tick cement, while others are playing some other yet undetermined role in tick feeding.     

The influence of various factors on fluid secretion by in vitro salivary glands of ixodid Ticks.

1. Salivary glands of the female ixodid tick, Dermacentor andersoni, secrete fluid in vitro when bathed in a slightly modified version of the mammalian tissue culture medium 'TC 199'. 2. Rate of salivation in vitro increases with progression of feeding, but there is no comparable increase in dry weight of the salivary glands during the early phase of engorgement. Engorged ticks secreted at only 25% the rate of 90-250 mg ticks, indicating that salivary gland degeneration has already begun in the very early post-engorgement stage. 3. A salivary gland stimulating factor can be detected in the nervous system but not in other tissues. 4. Male salivary glands secrete at only 1/20th the rate of female glands. Thus males probably do not use their salivary glands as osmoregulatory organs. 5. From the uniform lack of response to ACh and uniform response to DA in 7 ixodid tick species, it is suggested that the control of salivation is similar throughout the ixodid family.     

Female tick Hyalomma marginatum marginatum salivary glands: preliminary study on protein changes during feeding process and antigens recognized by repeatedly infested cattle

Proteins extracted from salivary glands of unfed, three days and five days fed adult Hyalomma marginatum marginatum were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). We have noticed changes during the three feeding steps. Some proteins disappeared during feeding process (23, 38, 39, 40 to 50, 95 and 112 kDa), they might be proteins which were converted in other substances and are secreted. Other antigens (13 to 14, 20, 25, 29, 165 and 210 kDa) were synthesized as a result of tick attachment and feeding. They may be related to growth and development or are the ciment which fixed the adult. Also, three Holstein calves were infested five times with 100 pairs of adult ticks of the same species. The five infestations were performed two weeks from the previous infestation. The sera before infestations and after each infestation were used in western-blot analyses to identify antigens from five days salivary gland extracts of the primary infestation of ticks. Three antigens (18.7, 50 and 80 kDa) were revealed weakly after the first and the second infestations by sera samples but not at infestation onward. Others (13.5, 17 to 18.5, 25, 30, 70, 133, 176 and 193 kDa) were revealed only by sera taken after manifestation of resistance (third infestation). A 13.5 kDa antigen was particularly revealed when resistance had appeared and became more evident after the fourth and fifth infestations. The late antigens recognized might be associated with establishment of calves resistance against ticks.     

Immunology of interactions between ticks and laboratory animals

There is good reason to believe that the resistance to ixodid ticks acquired by guinea pigs, rabbits and mice is immunologically mediated. One proposed mechanism for this resistance, which may well be common to all these laboratory animals, involves cutaneous hypersensitivity reactions. Basophils accumulate at tick attachment sites in the skin of resistant animals and degranulate in response to tick salivary antigens, releasing histamine and other mediators. The mediators may directly cause ticks to cease salivating and feeding and then to detach, or they may induce reflex grooming reactions by the host, leading to the removal of ticks from the itching skin.There are gaps in the evidence supporting this hypothesis, and it is likely that other modes of tick resistance remain to be described. However, it should be recognized that, although there have been a few details added to the story in the last fifty years, William Trager's original classic observations and conclusions still stand as the core of the current dogma.     

A cement protein of the tick Rhipicephalus appendiculatus,located in the secretory e cell granules of the type III salivary gland acini,induces strong antibody responses in cattle

Protein components of the cement cone of ixodid ticks are candidates for inclusion in vaccines against tick infestation, since they are essential for tick attachment and feeding. We describe here the cloning of a cDNA encoding a 36 kDa protein, designated Rhipicephalus Immuno-dominant Molecule 36 (RIM36), present in salivary glands and the cement cone material secreted by Rhipicephalus appendiculatus. The 334-amino-acid sequence of RIM36 has a high content of glycine, serine and proline. The protein contains a predicted N-terminal signal peptide and two classes of glycine-rich amino acid repeats, a GL[G/Y/S/F/L] tripeptide and a GSPLSGF septapeptide. Comparison of genomic and cDNA sequences reveals a 597 bp intron within the 3' end of the RIM36 gene. Immuno-electron microscopy demonstrates that RIM36 is predominantly located in the e cell granules of the type III salivary gland acini. An Escherichia coli recombinant form of the proline-rich C-terminal domain of RIM36 reacts with antisera from Bos indicus cattle, either experimentally infested with R. appendiculatus, or exposed to ticks in the field. The 36 kDa protein is strongly recognised on Western blots of salivary gland lysates and soluble extracts of purified R. appendiculatus cement cones by polyclonal antibodies generated against recombinant RIM36, and by antisera from cattle experimentally infested with ticks. The data indicate that this tick cement component is a target of strong antibody responses in cattle exposed to feeding ticks.     

Salivary glands in ixodid ticks: control and mechanism of secretion

The salivary glands are vital to the biological success of ixodid ticks and the major route for pathogen transmission. Important functions include the absorption of water vapor from unsaturated air by free-living ticks, excretion of excess fluid for blood meal concentration, and the secretion of bioactive protein and lipid compounds during tick feeding. Fluid secretion is controlled by nerves. Dopamine is the neurotransmitter at the neuroeffector junction regulating secretion via adenylate cyclase and an increase in cellular cAMP. Dopamine also affects the release of arachidonic acid which is subsequently converted to prostaglandins. Prostaglandin E(2) (PGE(2)) is secreted at extremely high levels into tick saliva for export to the host where it impacts the host physiology. Additionally, PGE(2) has an autocrine or paracrine role within the salivary gland itself where it interacts with a PGE(2) receptor to induce secretion (exocytosis) of bioactive saliva proteins via a phosphoinositide signalling pathway and an increase in cellular Ca(2+). Regulation of fluid secretion has been extensively studied, but little is known about the mechanism of fluid secretion. Continuing advances in tick salivary gland physiology will be made as key regulatory and secretory gland proteins are purified and/or their genes cloned and sequenced.     

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.     

Antigens of Amblyomma americanum ticks recognized by repeatedly infested sheep

Sera were taken from 3 sheep that had been infested 5 times with Amblyomma americanum and that exhibited manifestations of humoral depression to homologous antigens and anti-tick resistance. Proteins extracted from the intestine or salivary glands of unfed ticks or salivary glands from partially (3-day) fed ticks were analyzed by polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-PAGE. Antigens recognized by the sheep in the same materials before and after each infestation were analyzed by western blots. The sheep responded to 44 antigens. Nine to 23 antigens were recognized by the preinfestation sera and the sera of 2 gnotobiotic sheep. Four antigens (34,000, 36,500, 38,000, and 115,000 MW) were revealed conspicuously by the serum of the first infestation but very weakly or not at all by the sera of the third infestation onward. Two antigens (35,500 and 29,000 MW) from fed salivary glands were revealed only by sera taken after manifestations of resistance had appeared. These antigens may be responsible for anti-tick protection. The 29,900 MW antigen was present also in salivary extracts of Boophilus microplus.     

Molecular cloning of cAMP-dependent protein kinase catalytic subunit isoforms from the lone star tick, Amblyomma americanum (L.)

The salivary glands of ixodid ticks are central to tick feeding and to survival during off-host periods. They produce and secrete a number of molecules critical to maintaining the complex host-vector interface and to maintaining osmotic balance. We have previously shown that a cyclic AMP-dependent protein kinase (cAPK) is involved in the mechanism of salivary gland secretion. We have now cloned cDNAs encoding three isoforms of the catalytic subunit (cAPK-C) of the cAPK from Amblyomma americanum, which are probably produced from alternative RNA processing of a single cAPK-C gene. The cDNAs contain unique N-termini of variable lengths that are linked to a common region containing the alpha A helix, catalytic core, and a C-terminal tail. The common region is highly similar to both insect and vertebrate cAPK-Cs. We have examined mRNA profiles in whole ticks and in isolated salivary glands throughout feeding and find that a single cAPK-C isoform is expressed in the salivary glands of both unfed and feeding females.     

An immunocytochemical marker for the complex granules of tick salivary glands which traces e-granule shedding to interstitial labyrinthine spaces

An antigenic protein (12C), previously isolated from salivary glands of Rhipicephalus appendiculatus, as a possible factor in host resistance to repeated tick infestation, is labeled by protein A-gold immunocytochemistry in adult feeding ticks. It is a component of the gland's complex a-, d- and e-granules and also appears within the chitinous walls of intercalated ducts. In females, between days 4 and 7 of feeding, labeled e-granules appear also within the labyrinthine spaces of acinus type III, apparently released from e-cells as the abluminal interstitial cells initiate formation of a basolateral labyrinth. Granules thus shed are fragmented by interstitial cell processes, some fragments being phagocytized, others disintegrating to single point label scattered throughout the labyrinth. The latter possibly may pass into the acinar lumen. By the eighth day the label is gone from the labyrinth.