Tick saliva: recent advances and implications for vector competence

Medical and Veterinary Entomology (1997) 11,277-285

Tick saliva: recent advances and implications for vector competence A . S . B O W M A N , L . B . C O O N S , * G . R . N E E D H A M t and J . R, s AUE R Department of Entomology, Oklahoma State University Stillwater, Oklahoma, *Department of Microbiology and Molecular Cell Sciences, University of Memphis, Memphis Tennessee, and +Acarology Laboratory, Department of Entomology, The Ohio State University, Columbus, Ohio, U.S.A.

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 secretions with special emphasis on how they favour pathogen transmission.

Key words. Ixodidae, tick, saliva, pathogen, vector, anti-platelet, haemostasis, anticoagulant, vasodilator, immunomodulation, anti-inflammatory.

Introduction

Several attributes favour the success of ticks as ectoparasites. Not the least is a striking resiliency off the host where the tick can survive many months without taking a bloodmeal (Needham & Teel, 1991). Conversely, after attachment to a host, ixodid ticks ingest a copious meal of blood (females typically imbibe more than 100 times their unfed body weight) during a prolonged attachment of 7-15 days. The morphological and physiological adaptation of the ixodid tick to survive off the host for extended periods precludes it from rapidly engorging as do haemophagous insects. Therefore, a prolonged attachment period is required for the tick to become adapted for haematophagy and be able to accommodate the many-fold increase in body size. Protracted attachment and feeding is possible largely because of properties of the tick’s saliva which

Editorial footnote: This refereed review was first presented verbally at a symposium on ‘Anthropod-Host-Pathogen-Interaction’ at the XX International Congress of Entomology, Florence, August 1996.

Correspondence: Dr Alan S. Bowman, Department of Entomology, 127 Noble Research Center, Oklahoma State University, Stillwater, OK 74078, U.S.A.

counter host immune, inflammatory and haemostatic reactions at the feeding site. There is increasing evidence that factors in the saliva favouring successful tick feeding also favour establishment of pathogens in the host (Jones etal., 1990; Zeidner etal., 1996). In the present review we discuss current information and concepts on tick salivary secretions, with emphasis on their role in promoting successful tick feeding. Details of salivary gland morphology and physiology are in Sauer et al. ( 1 995) and earlier literature on tick-host interactions in Kaufman (1989) and Ribeiro (1987). The significance of prostaglandins in tick saliva is discussed by Bowman et al. (1 996). For general information about tick biology the reader is directed to the work by Soneshine (1991).

Secretory products

Water and ions

The saliva of ixodid ticks consists mostly of water and ions. This is because ixodid ticks concentrate the bloodmeal while the tick is still attached and feeding and secrete the excess fluid back to the host via the salivary glands. The large increase in tick weight noted above that occurs during tick feeding therefore

0 1997 Blackwell Science Ltd 277

278 A. S. Bowman etal.

greatly underestimates the total volume of blood imbibed. Various estimates indicate that 33-50% of all fluid ingested is excreted back to the host via this unique mechanism (Sauer & Hair, 1972; Kaufman & Phillips, 1973; Koch & Sauer, 1984). The female lone star tick, Amblyomma americanum (L.) (Acari: Ixodidae), increases from about 4mg to 600mg during 9-1 5 days feeding, approximately 1 ml of blood is imbibed and 0.41111 of fluid excreted via the salivary glands.

Cement

Firm attachment of ixodid ticks to the host is enhanced by an attachment cement produced in the type I1 and I11 alveoli of the salivary glands (Jaworski et al., 1992). Ixodid tick genera that penetrate the epidermis superficially, e.g. Boophilus spp., secrete an external cement cone whereas others, e.g. Amblyomma spp., that penetrate the host more deeply secrete a cement casing around the mouthparts (Moorhouse, 1969; Kemp et al., 1982). The cement is largely proteinaceous but it also contains substantial amounts of lipid and glycoproteins (Kemp et al . , 1982). A 90kDa protein stimulates an immune response in the host and appears to be a common constituent of cement in both primitive deeply attaching species (e.g. Arnblyomma, Ixodes) and more evolved ticks that attach more superficially (e.g. Dermacentoc Rhipicephalus) (Jaworski et al..f992).

Anti-haernostatic factors

After attaching to the host, a feeding lesion or cavity forms in the host dermis by a complex series of events that involve both the tick and the host (Binnington & Kemp, 1980). Small blood vessels are ruptured and drain into the lesion. Such blood flow normally is arrested rapidly and completely by the hosts’ haemostatic processes. These mechanisms consts of (i) platelet aggregation to form a haemostatic plug that blocks the puncture hole in the blood vessel, (ii) activation of the plasma coagulation cascade to form a fibrin-mesh that holds the platelet plug in

place, and (iii) vessel contraction that can seal the injury site. The size of the blood vessel injured determines the relative importance of each of the three haemostatic processes. For the small dermal blood vessels ruptured during tick feeding the platelet plug and vasoconstriction should be adequate to cause cessation of the blood flow into the feeding lesion. The saliva of ticks contains factors that block the haemostasis at all three points by inhibiting (i) platelet adhesion, activation and aggregation, (ii) blood coagulation, and (iii) vasoconstriction and promote vasodilation.

Antiplateletfactors. A major salivary anti-platelet aggregation factor in blood-feeding arthropods is reported to be apyrase (Ribeiro, 1987), an enzyme that hydrolyses the adenosine diphosphate (ADP) released from injured cells and activated platelets. Apyrase has been found in the saliva of Ixodes dammini Spielman et al. (Ribeiro et al., 1985) [Ixodes dammini = I,scapularis (Oliver et al., 1993)] and Ornithodorus moubata (Murray) (Ribeiro et al., 1991); however, apyrase appears to be absent from the saliva of A.americanum (J. M. C. Ribeiro, pers. comm.).

In addition to the salivary apyrase activity, Omoubata produces three highly potent factors that interfere with platelet function. Moubatin, a 17 kDa polypeptide isolated from whole tick homogenates, potently inhibits collagen-dependent platelet aggregation (IC5(, = 5 0 n ~ ) , but was ineffective against aggregation induced by thrombin, arachidohic acid or A23 187, and was only effective against ADP-induced aggregation at higher concentrations (Waxman & Connolly, 1993). The amino acid sequence of moubatin shows little homology to any other protein and lacks the Arg-Gly-Asp sequence which is a characteristic of other platelet-aggregation inhibitors belonging to the disintegrin family. A small peptide (6 kDa), termed disagregin, from Omoubata salivary glands, inhibits ADP-induced platelet aggregation (ICs,, = 104nM), as well as inhibiting aggregation due to collagen, thrombin, adrenaline and platelet activating factor (Karczewski et al. , 1994). Disagregin inhibits platelet aggregation by binding to the platelet membrane fibrinogen receptor GIIb-IIIa and, hence, also inhibits platelet adhesion to fibrinogen. Though disagregin shows many functional

Table 1 . Characteristics of some recently identified anti-platelet aggregatory activities in tick salivary glands and saliva.

Peptide Species Compound Target weight Reference

0.moubata 0.moubata 0.moubata

0.moubata

D. variabilis

1.dammini 1.dammini A.americanum

Apyrase Moubatin* Disagregin

Tick Adhesion Inhibitor Variabilin

Apyrase PGI, E D 2

ADP Unknown Fibrinogen receptor (GIIb-IIIa) Collagen receptor (Gla-IIa) Fibrinogen receptor (GIIb-IIIa) ADP PG1,-receptor PGI,/D,-receptor

Unknown 17 kDa 6 kDa

15kDa

5 kDa

Unknown NA NA

Ribeiro etal., 1991 Waxman & Connolly, 1993 Karczewski et al., 1994

Karczewski et al., 1995

Wang et al. , 1996

Ribeiro et al., 1985 Ribeiro et al., 1988 Bowman ef al., 1995

~

* Moubatin was isolated from homogenates of whole ticks. Its presence in the salivary glands has not been examined.

0 1997 Blackwell Science Ltd, Medical and Veterinary Entomology 11: 277-285

Tick saliva 279

properties with members of the disintegrin family, it does not possess the Arg-Gly-Asp recognition sequence. In addition, a peptide ( 1 5 kDa) that blocks the adhesion of platelets (1c5,) = 8nM) to a collagen-coated microtitre plate has been identified in the salivary gland of Omoubata (Karczewski et al., 1995) and has been termed Tick Adhesion Inhibitor (TAI). TAI appears to mediate its inhibitory effect by binding to the platelet membrane at the glycoprotein Ia-IIa complex (the platelet VLA-2 integrin complex) at the site involved in the initial attachment to collagen. It would appear that 0.moubata possesses several compounds capable of preventing platelet activation (Table 1) by nullifying the effect of (i) exposed collagen on damaged blood vessel walls (moubatin, TAI, disagregin); (ii) fibrinogen formed (disagregin); ( i i i ) ADP formed (apyrase, disagregin); and (iv) any thrombin formed (disagregin, anticoagulants (see below)).

A small peptide (5 kDa), named variabilin, has recently been isolated from Dermacentor variabilis (Say) salivary glands and inihibits (ICs0 = 157 n ~ ) ADP-induced platelet aggregation (Wang et al., 1996). Variabilin, like disagregin from O.moubata, inhibits platelet aggregation due to xcits antagonism at the fibrinogen receptor GPIIb-IIIa. However, unlike disagregin, variabilin does contain the Arg-Gly-Asp recognition sequence common to most of the disintegrin family, though variabilin exhibits little sequence homology to any other known protein (Wang et al., 1996).

Tick salivary glands also secrete large amounts of prostaglandins (Bowman et al., 1996). Both prostacyclin (PGI,) and PGD, prevent platelet aggregation by inhibiting platelet secretion of ADP and have been found in the saliva of I.dammini and A.americanum respectively (Ribeiro etal., 1988; Bowman etal., 1995). PGI, is the most powerful known inhibitor of platelet aggregation, acting at concentrations of <I ng/ml and can actually cause disaggregation of those platelets that have aggregated (Radomski et al., 1987). Ixodes dammini saliva contains PGI, at 523ng/ml (Ribeiro et al., 1988), and thus is capable of inhibiting platelet aggregation at the feeding lesion. Overall, tick saliva contains a diverse repertoire of factors with the potential for inhibiting platelet aggregation at the feeding site. The number of factors in the tick’s saliva against platelet aggregation matches the complexity of platelet activation and is probably a good indication of the importance of platelets to successful tick feeding.

Anticoagulant factors. Blood coagulation can occur through the intrinsic and extrinsic pathways and involves a series of proteolytic reactions that ultimately lead to the thrombin- catalysed conversion of soluble fibrinogen to an insoluble fibrin mesh that holds the platelet plug in place. The intrinsic pathway is triggered by exposure of blood to subendothelial components, such as collagen, following trauma. The extrinsic pathway is initiated by the release of tissue thromboplastin from injured cells. Though the intrinsic and extrinsic pathways are inde- pendent and have separate factors in their initial cascade, the activated serine protease factor Xa (ma) is common to both pathways and, thus, plays a pivotal role in coagulation (Fig. 1). It is the action of fXa on prothrombin that generates thrombin, an important protease, that not only is responsible for the formation of the fibrin mesh, but also is an important factor in platelet stimulation (see below). Potent anticoagulant activities have been reported in the saliva or salivary glands from a dozen

IX

Tick species: 10 1

+ Xa\ ITick species: 1- 8 I

L prothrombin -thrombin I

C-3d Tick species: 2- 91

/L platelet fibrinogen - fibrin aggregation (clot)

Flg. 1. A simplified diagram of the clotting cascade showing the enzymatic points at which anticoagulants from different tick species exert their inhibition. Tick species: ( 1 ) R.appendiculatus; (2) Ornirhodoms moubata; (3) Amblyomma americanum; (4) Hyalomma truncatum; ( 5 ) Ixodes holocyclus; (6) Boophilus microplus; (7) Haemaphysalis longicomis; (8) Omithodoms savignyi; (9) Ixodes ricinus; (10) Dermacentor andersoni (See Table 2 for appropriate references.)

tick species, both hard and soft ticks. With the exception of Dermacentor andersoni Sriles (Gordon & Allen, 1991), all the tick anticoagulants studied to date inhibit either fXa or thrombin induced coagulation (Fig. 1, Table 2).

Tick anticoagulants are heat inactivated and susceptible to protease deactivation. The anticoagulants are relatively stable proteins, an obvious advantage for a compound to be secreted into a foreign environment and still maintain its activity, but the anticoagulant from Ornithodoms savignyi(Aud0uin) is unusually robust. Salivary gland extracts from Omvignyi could undergo freeze-thaw cycling, sonication, be heated to 95°C in the presence of reducing agents, subjected to electrophoresis including membrane blotting, treated with acetone and dried, and this material still maintained potent anticoagulant activity (Gaspar et al., 1995). The tick anticoagulants are all small (7-17kDa, see Table 2), except for the activity in the salivary glands of Rhipicephulus appendiculatus (Neumann that is 65 kDa (Limo et al., 1991). The R.appendiculatus anticoagulant inhibited ma-induced clotting ((Limo et al., 1991). However, this anticoagulant did not inhibit fXa activity on a synthetic substrate, leading the authors to conclude that the anticoagulant acts at a site distinct from the active site of fXa (Limo et al., 1991). Other anti-fXa activities found in the salivary glands of different tick species inhibit the action of purified fXa on synthetic substrates,


280 A. S. Bowman et al.

Table 2. Characteristics of some recently reported anticoagulants in tick salivary glands and saliva.

Molecular Species Source Target weight Reference

0.moubata R.appendiculatus H. rruncatum I . holocyclus 3. niicmplus H.1ongicornis 0.savignyi A.americanum 0.moubata I . ricinus D. anderson i

Whole tick Salivary gland Salivary gland Salivary gland Salivary gland Salivary gland Salivary gland Saliva Whole tick Whole tick Salivary gland

m a fXa fXa fXa and thrombin fXa and thrombin fXa and thrombin fXa and thrombin fXa and thrombin Thrombin Thrombin N and Nll

7 kDa 65 kDa I7kDa NA NA NA I4 kDa 16 kDa I7kDa 7 kDa

NA

Waxman er al., 1990 Limo et al.. 1991 Joubert ef al., 1995 Anastopoulos et al., 1991 Anastopoulos et al., 1991 Anastopoulos et al., 1991 Gaspar er al., 1995 Zhu er al., 1997 Waxman & Connolly, 1993 Hoffman etal . , 1991 Gordon & Allen, 199 I

NA = not reported.

indicating that these anticoagulants do act by inhibiting the amidase activity site of fXa (Waxman e t al., 1990; Anastopoulos et al., 1991 ; Gaspar et al., 1995; Joubert et al., 1995; Zhu et al., I997a). Thrombin inhibitors have been reported in Omoubata and A.americanum where thrombin-dependent clotting of plasma was inhibited by salivary gland extracts (Waxman & Connolly, 1993; Zhu e t al., 1997a) and also in i xodes ricinus L., Boophilus microplus, (Canestrini), ixodes holocyclus Neumann, Haemaphysalis longicornus Neumann and 0.savignyi where thrombin activity against synthetic chromogenic substrates was inhibited (Anastopoulos et al., 1991; Gaspar et al., 1995).

By far the best characterized tick anticoagulant is tick anticoagulant peptide (TAP), a serine protease inhibitor specific for fXa from the soft tick 0.moubata. It should be noted that the TAP was originally isolated from whole 0.moubata homogenates (Waxman et al . , 1990), and all subsequent work has been performed on recombinant TAP. Hence, although suspected to originate from the salivary glands, the actual site of TAP production is still unknown. TAP is a single-chain acidic polypeptide composed of sixty amino acids with limited homology to the Kunitz-type inhibitors (Waxman et al., 1990). A recombinant version of TAP (rTAP) expressed in a number of systems has allowed production of large enough quantities for many detailed studies, a limiting factor in studying the anticoagulants from other tick species. rTAP is a reversible, slow, tight-binding inhibitor of fXa displaying a competitive type inihibition and apparently involving the active site of fXa (Jordan et al., 1990). Later studies showed that rTAP was a 30-fold more potent (K, = 5 . 3 ~ ~ ) inhibitor of fXa when tested in the presence of the prothrombinase complex co-factor N a , suggesting that the interaction between fva and fXa cause a conformational change in fXa which makes it more vulnerable to rTAP inhibition (Krishnaswamy et al., 1994).

It is 100 years since a tick anticoagulant was first reported (Sabbatini, 1898), and many studies have been done recently in the field of tick anticoagulants. However, the functions of these anticoagulants have not been fully addressed. The obvious role of anticoagulants is the prevention of the host blood from coagulating. However, this may be a rather simplistic assump- tion. The importance of clot formation in haemostasis in the microcirculation is unclear. In animals with marked impairment

in either the intrinsic or extrinsic pathway of coagulation, a platelet plug still readily forms when a vessel in the microcirculation is cut, and this platelet plug is similar in mass to that found in normal animals (Hovig et al., 1967). One reason why ticks produce these anticoagulants may be that ticks are not so much concerned with thrombin activity as it relates to fibrin production and clot stabilization of the haemostatic plug, but rather the role that thrombin plays in platelet stimulation. Thrombin is the most potent platelet aggregating agent (Packham, 1994). Platelet aggregation could be thwarted by either inhibiting the formation of thrombin (i.e. anti-ma) or inhibiting the enzymatic activity of thrombin (i.e. antithrombin). Tick salivary glands appear to secrete both fXa and thrombin inhibitors in preference to targeting factors elsewhere in the coagulation cascade (Fig. 1). Further supportive evidence of this hypothesis is that mosquitoes also contain specific fXa and thrombin inhibitors (Stark & James, 1995, 1996a), though the kinetics of coagulation are longer than the time required for a mosquito to obtain a bloodmeal (Stark & James, 1996b).

A valid alternative hypothesis for the role of anticoagulants in tick feeding is not to inhibit coagulation at the feeding site, but rather to maintain fluidity of the blood as it passes through the mouthparts and into the gut. The level of anticoagulant activity in the salivary glands (Gordon & Allen, 1991) or saliva (Zhu er al., 1997a) increases greatly as the tick enters its rapid phase of feeding when the majority of the bloodmeal is ingested over a short period of time. This temporal pattern of anticoagulant production would be consistent with a role for the anticoagulants in preventing coagulation in the gut and hindering movement of blood around the gut. Indeed, far greater anticoagulant activity was detected in homogenates of 1. holocyclus, Bmicroplus and H. fonigcomus from which the salivary glands had been removed than in the salivary gland homogenate themselves (Anastopoulos et al., 1991). These authors suggested that the anticoagulant in the saliva is injected into the attachment site and then ingested with the bloodmeal, though, intuitively, it would seem to be more efficient for the gut tissues themselves to secrete the anticoagulant.

Anti-vasoconstrictors and vasodilators. Although vasoconstriction alone appears to be inadequate to cause haemostasis without the participation of platelets and the coagulation system

0 1997 Blackwell Science Ltd, Medical and Veterinary Enfomology 11: 277-285

Tick saliva 28 1

for larger vessels, vessel contraction may be sufficient to seal the injury site in the microcirculation (Mustard & Packham, 1977). Potent vasoconstricting peptides, the endothelins, are released by injured vascular endothelium and cause extreme local vasoconstriction (Silldorf et al., 1995). Vasoconstriction is also induced by thromboxane A, and serotonin secreted by activated platelets. As already discussed, tick saliva contains several compounds to inhibit platelet activation. It is thought that tick saliva not only prevents vasoconstriction but is also capable of causing vasodilation, thus allowing more fluid to enter the feeding lesion. Though extremely potent vasodilatory peptides have been reported in the salivary glands of several haemotophagous insects, to date, the only vasodilatory compounds reported in tick saliva are prostaglandins (see Bowman etal., 1996). PGI,, PGE,, and to a lesser extent PGD, are potent vasodilators. PGE, and PGI, have been identified in the saliva of I.dammini (Ribeiro et al., 1985, 1988), and PGE, and PGD, in the saliva of A.americanum (Ribeiro et al., 1992; Bowman et al., 1995). The highest level of PGE,, in the salivary glands of Bmicroplus was just prior to the tick entering the rapid feeding phase (Dickinson et al., 1979) when increased blood flow to the feeding lesion would seem most advantageous. In addition, when the prostaglandin-producing capability of ticks was impaired by dietary alteration of the host blood lipids the engorged tick weight was reduced by 30% (Madden et al., 1996).

Immunornodulation

Natural hosts infested with ticks are characterized by greatly reduced or absent immune responses (Trager, 1939; Riek, 1962; George et al., 1985). Recent work has begun to elucidate factors present in the tick saliva that result in this immunosuppression. Complement, antibody production, cytokine elaboration and proliferative responses of host T-lymphocytes are known to be suppressed by factors in tick saliva in vitro (Wikel et al., 1994). Although prostaglandins in tick saliva are likely candidates for contributing to immunosuppression in the host (Bowman et al., 1996), increasing evidence indicates that protein factors in tick saliva are also important (Wikel, 1996). This topic is reviewed in detail by Brossard & Wikel(1997).

Saliva of I.dammini inhibits the alternative pathway of complement activation (Ribeiro, 1987) and anaphylatoxin activity (Ribeiro & Spielman, 1986). Salivary gland extracts from partially fed D.andersoni females inhibited mice and cattle lymphocyte responsiveness to concanavalin A and enhanced lymphocyte proliferation in response to LPS (Ramachandra & Wikel, 1992, 1995). Salivary gland extracts also inhibited macrophage elaboration of proinflammatory cytokines JL-I and TNF-a, and T,1 -lymphocyte secretion of IL-2 and INF-)I (Ramachandra & Wikel, 1992).

Little is known about the identity or mode of action of factors in tick saliva causing immunosuppressive effects in the host. Lipid soluble and detergent soluble fractions from salivary glands of Dmdersoni were found to suppress concanavalin A- induced blastogenesis of murine splenocytes (Bergman et al., 1995). Salivary gland preparative SDS-PAGE fractions containing one or more soluble proteins with molecular weights from 36 to 43 kDa suppressed the splenocyte responses to concanavalin

A. Inokuma et al. (1994) indicated that inhibition of lymphocyte responsiveness to PHA was due to prostaglandin E, in the saliva of Bmicroplus. A salivary protein from I.dammini having a molecular weight of greater than 5 kDa inhibited proliferative responses of mouse splenocytes to concanavilin A and PHA via a non-PGE,-dependent mechanism (Urioste etal., 1994). Ribeiro ( 1 987) identified a 49 kDa protein in the salivary glands of I.dammini that inhibits the alternative complement pathway.

Because the inflammatory and immune systems are intimately linked, the salivary components discussed above that exert inhibitory effects upon the cells of the immune system, especially the macrophage, are also responsible for the lack of inflammatory response. A few activities more specifically pertaining to the inflammatory response have been identified in tick saliva or salivary glands. Neutrophils are particularly important in the inflammatory response and their function is inhibited in the presence of [.dammini saliva (Ribeiro et al., 1990). Histamine is a potent mediator of the inflammatory response and the salivary glands of Rhipicephalus sanguineus (Latrielle) are reported to possess an antagonist of histamine (Chinery & Ayitey-Smith, 1977). A kininase activity is present in I.dammini saliva (Ribeiro, 1987) that is capable of deactivating bradykinin, another inflammatory mediator that potentiates pain. The high levels of prostaglandins in tick saliva have been suggested to inhibit the inflammatory response (Bowman et al., 1996) by inhibiting neutrophil function and suppressing the release of inflammatory mediators from mast cells.

Enzymes and other proteins

A striking feature of the female ixodid salivary gland is its remarkable growth, differentiation, development and sequential accumulation and depletion of materials within specific cells during tick feeding (Binnington, 1978). Consequently, differential gene expression occurs in the salivary glands at different stages of tick feeding and salivary gland development (Oaks etal., 1991). A different and more numerous m a y of proteins is obtained in the dopamine-induced saliva from female A.americanum during the early part of the feeding phase than during the later phase (McSwain et al., 1982). Many different activities in tick saliva have been reported (for review see Neitz et al., 1987; Sauer et al., 1995), but here we discuss some intriguing salivary components described more recently.

It has long been known that host immunoglobulin G (IgG) in the bloodmeal can traverse the gut into the haemolymph (Ben-Yakir et al., 1986). Recently it was shown that host IgG was present in the saliva at 10-fold the concentration found in the haemolymph in R.appendiculatus and that this IgG retained its binding activity to specific antibodies (Wang & Nuttall, 1994). Several specific IgG binding proteins (IGBPs) were detected in the haemolymph and these were different from the IGBPs detected in the salivary gland (Wang & Nuttall, 1995a). Host IgG directed against tick antigens could damage host tissues if allowed to freely circulate in the haemocoel. It was suggested that the IGBPs present in the haernolymph and salivary glands work in concert to bind, transport and finally excrete host IgG back into the host via the saliva (Wang & Nuttall, 1994). The idea that ticks might possess a complex and multi-component



mechanism for neutralizing and excreting potentially harmful host IgG is intriguing when protease deactivation or binding proteins at the gut wall interface would appear to be a simpler method. Wang & Nuttall (1995b) suggested that perhaps the high IgG levels in the saliva secreted back into the host serve to dampen the host’s immune response by interacting with the Fc receptors on various immune cell types at the feeding lesion, though it is unclear if intact IgG orjust the antigen binding fragment (Fab) is present in the IGBP-IgG complex. Because much work toward anti-tick vaccines is directed against intrahaemocoelic targets needed to be accessible to IgG circulating in the haernolymph, elucidation of how ingested host IgG is processed by ticks is of considerable importance.

A phospholipase A, has been found in the saliva of A.americanum that has a molecular weight of 55kDa as determined by size exclusion chromatography (Bowman et al., 1997). Because extracellular PLA,s, such as found in pancreatic secretions, bee and snake venoms, are all 14Da, the 55kDa PLA, found in tick saliva is most unusual. In addition, the tick salivary PLA, is activated at micromolar calcium levels which is a characteristic for intracellular PLA,s, not extracellular PLA2s which are activated at a thousand fold higher calcium level. The function of this novel PLA, is unknown. Recently a heat- and trypsin-sensitive haemolytic activity was identified in the saliva of Aamericanum (Zhu et al., 1997b). Size exclusion chromatography of saliva proteins revealed one peak of activity that correlated with the activity of tick saliva phospholipase A, (PLA,) activity both having the molecular weight of 55 kDa. The haemolytic activity was Ca2+-dependent and inhibited by the PLA, inhibitor oleyloxyethyl phosphorylcholine. The activity is postulated to lyse host red blood cells at the feeding site, thus facilitating the tick digestive process (Zhu, et al., 1997b).

Calreticulin (CRT), a multi-functional Ca2+-binding protein, is present in the salivary glands and saliva of female A.americanum and D.variabilis (Jaworski ef al., 1995). CRT is a ubiquitous protein that is localized in the endoplasmic reticulum of most animals where it plays a role in the control of intracellular Ca”. Most roles for CRT are intracellular. How- ever, tick-CRT does not have the usual endoplasmic reticulum retention signal (KDEL), but instead has a variant (HEEL). This variant may not anchor the protein as firmly as KDEL to the endoplasmic reticulum, making it less likely to be retained intracellularly, and might explain the occurrence of tick-CRT in the saliva. It is thought that tick-CRT originates from the type I11 acini of the salivary gland (Coons er al., in preparation). A function for tick-CRT is yet to be determined, but numerous events associated with bloodfeeding could be influenced by a Ca2+-binding protein, including coagulation, platelet aggregation and vasodilation.

Significance of tlck salivary secretory products to pathogen transmisslon

Pathogen development in ticks with specific reference to salivary glands has been reviewed recently (Sauer et al., 1995, 1996). Viruses, bacteria, rickettsia, protozoan and even nematode pathogens are all transmitted by ticks. Pathogens can be transmitted via coxal fluid in argasid ticks (Burgdorfer &

Hayes 1990), or possibly regurgitation in argasid and ixodid ticks (Brown, 1988; Connat, 1991). However, the most common route of transmission appears to be through the salivary glands (Ribeiro et al., 1987; Stitch et al., 1993; Kaufman & Nuttall, 1996) with replication of the pathogen in the salivary glands a common feature (Sauer et al., 1996). Interestingly, it was recently reported that saliva collected from ticks co-injected with Thogoto virus and a salivation stimulant contained Thogoto virus (Kaufman & Nuttall, 1996). This demonstrates that, at least for Thogoto virus, pathogens circulating in the haemolymph can rapidly be secreted in the saliva without any establishment in the salivary gland cells.

Little is known about how the tick’s physiology affects pathogen development inside the tick tissues, but it is becoming clear that secretory products have a tremendous impact upon the transmission and establishment of pathogens in the host. Ten-fold more uninfected Rappendicufatus became infected when feeding on a guinea-pig injected with Thogoto virus and tick salivary gland extract than if the virus was injected alone (Jones et al., 1989). The salivary activated transmission (SAT) factor was absent in extracts of other tissues in R.appendiculatus or in mosquito salivary glands, but was present in the salivary gland extracts of Amblyomma variegatum (Fabricius) (Jones et al., 1989). SAT activity was destroyed by protease treatment of salivary glands (Jones et al., 1990) indicating a proteinaceous nature, and it has been determined that SAT-factor occurs in the saliva as well as salivary gland extracts (Jones et al., 1992). It was postulated that the SAT-factor in tick saliva enhanced pathogen establishment by inhibiting the host’s immune response (Jones et al., 1992) and recent evidence supports this supposition. Tick salivary gland extracts have been shown to suppress in v i m production of the cytokines TNF-a, y-IFN and IL-2 (Ramachandra & Wikel, 1992). In an elegant study these two lines of research have converged. Mice infested with Borrelia burgdorferi infected. Ixapularis and simultaneously receiving peritoneal administration of TNF-a, yIFN and IL-2 exhibited up to 95% resistance to B,burgdorferi infection compared to control mice (Zeidner et al., 1996). Therefore it would appear that some tick salivary gland secretion suppresses the host immune response to the tick while inadvertently facilitating the establishment of tick-vectored pathogens.

Diversity of salivary factors: redundancy or necessity?

Platelets aggregation occurs within seconds and can cause haemostasis in the ruptured microcirculation independently of the other arms of the haemostatic system, namely coagulation and vasoconstriction. Not surprisingly, tick saliva counters platelet aggregation. The vertebrate platelet aggregatory system is complex and there are numerous ways by which platelets can be activated (e.g. ADP, thromboxane, thrombin, collagen, fibrinogen, adrenaline, serotonin, platelet-activating factor). In response, ticks have developed a similarly complex system in their saliva comprised of many factors affecting different aspects of platelet activation (Table 1). All these salivary anti-platelet aggregatory factors have the same ultimate goal to prevent the host platelets from being activated and aggregating, but they

0 1997 Blackwell Science Ltd. Medical and Veterinary Entomology 11: 277-285

Tick saliva 283

do so by distinctly different routes. For example, in the case of Omoubata, apyrase could thwart activation of platelets by ADP, but platelets coming into contact with collagen on damaged tissue would be activated without moubatin, whereas platelets coming into contact with fibrinogen require disagregin to prevent adhesion. Hence, there is no real evidence of over-lapping duplication or redundancy exhibited in the anti-platelet aggregatory factors in tick saliva. It is not known if all the activating pathways need be targetted, but A.arnericanurn saliva appears to lack apyrase (J.M.C. Ribeko, personal communication). Perhaps the platelets of some hosts are particularly sensitive to certain activators and, hence, ticks feeding predominantly on those hosts need to counter that particular activator. For example, the bird equivalent of platelets (thrombocytes) are insensitive to ADP, but aggregate in the presence of serotonin, collagen and thrombin (Belmarich et al., 1968). Tick species with a greater array of anti-platelet agregatory factors in the saliva may have a greater host range. Much may be learned in studies on tick salivary factors affecting haemostasis in several host systems.

In the case of countering the vertebrate coagulation system, there is more of an argument for overlapping duplication or possibly redundancy. The vast majority of species studied target either fXa or thrombin, and in many cases both factors (Table 2). It is suspected that in those cases where inhibition of only one factor has been reported, further work will reveal that both fXa and thrombin are inhibited. The primary role of fXa is the conversion of prothrombin to thrombin (Fig. 1). If fXa were inhibited negligible thrombin would be formed, whereas if thrombin were inhibited the coagulation cascade would be incomplete regardless of an active fXa. For example, the purified thrombin inhibitor from A.arnercinurn completely prevents whole sheep plasma from coagulating in vitm despite the presence of an optimum amount of active fXa (K. Zhu, personal communication). Why should so many tick species target both fXa and thrombin? There is amplification in the system, i.e. one molecule of active fXa may give rise to many hundreds of molecules of active thrombin. Therefore it would appear to be economical for the tick to produce enough anti-fXa to ensure total prevention of any thrombin production. However, complete fXa inhibition may be impossible in vivo and, hence, ticks provide the insurance of being able to inhibit thrombin. Using differing dilutions of purified fXa and thrombin inhibitors in whole blood clotting assays would reveal the relative importance of each inhibitor in an in vitro system and also any synergism between them. This approach may also reveal the relative importance of the thrombin inhibitor as an anticoagulant, as opposed to its role in platelet activation. Perhaps having the ability to inhibit coagulation via both fXa and thrombin may extend the host range by having the fXa-inhibitor tailored for one host species and the antithrombin designed for another. To date, tick anticoagulants have been tested against a limited number of typical laboratory animal models. Although there is a common goal for both fXa and thrombin inhibition in preventing coagulation, there is no real evidence that either inhibitor alone would suffice in vivo or, conversely, that either factor is superfluous. Therefore, if redundancy is taken to mean exceeding what is needed, the view that a degree of redundancy exists with regard to anticoagulants in tick saliva seems premature.

Summation and future outlook

Many tick attributes contributye to their efficiency of pathogen transmission including prolonged attachment to the host and copious salivation back into the host. It is by this transfer of saliva that various pharmacologically active compounds enter the host and suppress the host’s haemostatic, immune and inflammatory responses that would prevent the tick from completing its long bloodmeal. Saliva is also the oute by which most tick-borne pathogens are transmitted to the host and there is increasing evidence that components in the saliva enhance the transmission and establishment of pathogens in the host. There is a plethora of factors with pharmacological activity in tick saliva, but, at the same time, only a handful of molecules known to be present in tick saliva have been characterized and assigned a function. How these factors might interact together in vivo is an important issue, but models are not available that simulate the dynamic nature of the feeding site with multiple interacting host cell types in an increasingly pathological site.

It has been known for almost a century that tick saliva is rich in pharmacologically active components. Early pioneering research on Argaspersicus (Oken) salivary glands demonstrated inhibition of blood coagulation (Nuttall & Strickland, 1908) and that the toxin responsible for tick paralysis was present in the salivary glands of Ixodes holocyclus (Ross, 1926). Since that time, much work has been directed at elucidating the numerous activities in tick saliva, and in recent years research in this areas has grown at a rapid rate. The potential for some salivary components to be developed as therapeutic agents in the medical arena is receiving increasing attention from the pharmaceutical industry. As the disciplines of cell biology, molecular biology, immunology and biochemistry impinge upon the secretions of the tick salivary gland; one can expect that much new and exciting information is yet to emerge.

Acknowledgments

The authors express their thanks to Dr Rangappa Ramachandra and Dr Robert Barker for critically reviewing an earlier draft of the manuscript and to Dr Jose Ribeiro and Kuichan Zhu for helpful discussions. Approved for publication by the Director, Oklahoma Agricultural Experimental Station. Portions of the research reported herein were supported in part by NIH grant AI-31460 and AI-33665.

References

Anastopoulos, P., Thurn, M.J. & Broady, K.W. (1991) Anticoagulant in the tick Ixodes holocyclus. Australian Veterinary Journal, 68, 366- 367.

Belamarich, F.A., Shepro, D. & Kein, M. (1968) ADP is not involved in thrombin-induced aggragation of thrombocytes of a non-mammalian vertebrate. Nature, 220,509-5 10.

Ben-Yakir, D., Fox, J.C., Homer, J.T. & Barker, R.W. (1986) Quantitative studies of host immunoglobulin G passage into the hemocoel of the ticks Amblyomma americanum and Dermacentor variabilis. Morphology, Physiology and Behavioral Biology of Ticks (ed. by J. R. Sauer and J. A. Hair), pp. 329-34 I . Ellis Horwood Ltd, Chichester.

Bergman, D.K., Ramachandra, R.N. & Wikel, S . K . (l995)Dermucenror



andersoni: salivary gland proteins uppressing T-lymphocyte responses to concanavalin A in vitro. Experimental Parasitology, 81,262-27 1.

Binnington, K.C. (1978) Sequential changes in salivary gland structure during attachment and feeding of the cattle tick, Boophilus microplus. International Journal for Parasitology, 8.97-1 15.

Binnington, K.C. & Kemp, D.H. (1980) Role of tick salivary glands in feeding and disease transmission. Advances in Parasitology, 18,3 15- 339.

Bowman, A.S., Dillwith, J.W. & Sauer, J.R. (1996) Tick salivary prostaglandins: presence, origin and significance. Parasitology Today,

Bowman.A.S., Gengler, C.L., Surdick, M.R.,Zhu, K., Essenberg, R.C., Sauer, J.R. & Dillwith. J.W. ( I 997) A novel phospholipase A, activity in saliva of the lone star tick. Amblyomma americanum (L.). Experimental Parasitology (submitted).

Bowman, AS. , Sauer, J.R.. Zhu, K. & Dillwith, J.W. (1995) Biosynthesis of salivary prostaglandins in the lone star tick, Amblyomma americanum. Insect Biochemistry and Molecular Biology, 25, 735- 741.

Brossard. M. & Wikel, S.K. (1997) Immunology of interactions between ticks and hosts. Medical and Veterinary Enromo1og.v. 11, 270-276.

Brown, S. ( 1988) Evidence for regurgitation by Amblyoma americanum. Veterinaty Parasitology, 28, 335-342.

Burgdorfer. W. & Hayes, S.F. (1990) Vector-spirochete relationships in louse-borne and tick-borne borrelioses with emphasis on Lyme disease. Advances in Disease Vector Research, 6, 127-150.

Chinery, W.A. & Ayitey-Smith, E. (1977) Histamine blocking agent in the salivary gland homogenate of the tick Rhipicephalus sangineus. Nature, 265,366-367.

Connat, J.L. (1991) Demonstration of regurgiatation of gut content during blood meals of the tick Ornithodorus moubata: possible role in the transmission of pathogenic agents. Parasitology Research, 77,452454.

Dickinson, R.G., Binnington, K.C.. Schotz, M. & O’Hagan, J.E. (1979) Studies on the significance of smooth muscle contracting substances in the cattle tick Boophilus microplus. Journal of the Australian Entomological Society, 18, 199-210.

Gaspar, A.R.M.D., Crause, J.C. & Neitz, A.W.H. (1995) Identification of anticoagulant activities in the salivary glands of the soft tick, Ornithodoros savignyi. Experimental andAppliedAcarology, 19, I 17- 126.

George, J.E., Osborn, R.L. & Wikel, S.K. (1985) Acquistion and expression of resistance by Bos indicus x Bos taurus calves to Amblyomma americanum infestation. Journal of Parasitology, 71, 174-182.

Gordon. J.R. & Allen, J.R. (1991) Factors V and VII anticoagulant activities in the salivary glands of feeding Dermucentor andersoni ticks. Journal of Parasitology. 77, 167-170.

Hoffmann. A., Walsmann, P., Reisener, G., Paintz, M. & Markwardt, F. (1991) Isolation and characterization of a thrombin inhibitor from the tick lxodes ricinus. Pharmatie, 46. 209-2 12.

Hovig, T., Rowsell, H.C., Dodds, W.J., Jorgenson, L. & Mustard, J.F. (1967) Experimental hemostasis in normal dogs and dogs with cogenital disorders of blood coagulation. Blood, 30, 636-668.

Inokuma, H., Kemp, D.H. & Willadson, P. (1994) Prostaglandin, E, production by the cattle tick (Boophilus microplus) into the feeding sites and its effect on the response of bovine mononuclear cells to mitogen. Veterinary Parasitology, 53,293-299.

Jaworski, D.C., Rosell, R., Coons, L.B. & Needham, G.R. (1992) Tick (Acari: Ixodidae) attachment cement and salivary gland cells contain similar immunoreactive polypeptides. Journal of Medical Entomology

Jaworski, D.C., Simmen. F.A., Lamoreaux, W., Coons, L.B., Muller, M.T. & Needham, G.R. (1995) A secreted calreticulin in ixodid tick saliva. Journal of lnsea Physiology, 41, 369-375.

12,388-396.

29,305-309.

Jones, L.D., Hodgson, E. & Nuttall, P.A. (1989) Enhancement of virus transmission by tick salivary glands. Journal of General virology, 70,

Jones, L.D., Hodgson, E. & Nuttall, P.A. (1990) Characterization of tick salivary gland factor(s) that enhance Thogoto virus transmission. Archives of Virology, 1, (Suppl.) 227-234.

Jones, L.D.. Kaufman, W.R. & Nuttall, P.A. (1992) Modification of the skin feeding site by tick saliva mediates virus transmission. Experientia,

Jordan, S.P., Waxman, L., Smith, D.E. & Vlasuk, G.P. (1990) Tick anticoagulant peptide: kinetic analysis of the recombinant inhibitor with blood coagulation factor Xa. Biochemistry, 29, 11095- 1 IIOO.

Joubert, A.M., Crause, J.C., Gaspar, A.R.M.D., Clarke, F.C., Spicker, A.M., & Neitz, A.W.H. (1995) Isolation and characterization of an anticoagulant present in the salivary glands of the bont-legged tick, Hyalomma truncatum. Experimental and Applied Acarology, 19,

Karczewski, J. , Endris, R. & Connolly, T.M. (1994) Disagregin is a fibrinogen receptor antagonist lacking the Arg-Gly-Asp sequence from the tick. Ornrithodorus moubara. Journal of Biological Chemistry, 269, 6702-6708.

Karczewski, J., Waxman, L., Endris, R.G. & Connolly, T.M. (1995) An inhibitor from the argasid tick Ornithodoros moubata of cell adhesion to collagen. Biochemical and Biophysical Research Communications,

Kaufman, W.R. (1989) Tick-host interaction a synthesis of current concepts. Parasitology Today, 5.47-56.

Kaufman, W.R. &Phillips, J.E. (1973) Ion and water balance in the ixodid tick, Dermacentor andersoni. I. Routes of ion and water excretion. Journal of Experimental Biology, 58,523-536.

Kaufman, W.R. & Nuttall, P.A. (1996) Amblyomma variegatum (Acari: Ixodidae): mechanism and control of arbovirus secretion in tick saliva. Experimental Parasitology, 82.3 16-323.

Kemp, D.H., Stone, B.F. & Binnington, K.C. (1982) Tick attachment and feeding: role of the mouthparts, feeding apparatus, salivary gland secretions and the host response. Physiology of Ticks (ed. by F. D. Obenchain and R. Galun.), pp. 119-168. Pergamon Press, Oxford.

Krishnaswamy, S., Vlasuk, G.P. & Bergum, P.W. ( I 994) Assembly of the prothrombinase complex enhances the inhibition of bovine factor Xa by tick anticoagulant peptide. Biochemistry, 33,7897-7907.

Koch. H.G. & Sauer, J.R. (1984) Quantity of blood ingested by four species of hard ticks (Acari: Ixodidae) fed on domestic dogs. Annals of the Entomological Society ofAmerica, 77, 142-146.

Limo, M.K., Voight. W.P.,Tumbo-Oeri,A.G., Njogu, R.M. & Ole-Moiyoi, O.K. (1 99 I ) Purification and characterization of an anticoagulant from the salivary glands of the ixodid tick Rhipicephalus appendiculatus. Experimental Parasitology, 72,4 18-429.

Madden, R.D., Sauer. J.R., Dillwith, J.W. & B0wman.A.S. (1996) Dietary modification of host blood lipids affect reproduction in the lone star tick, Amblyomma americanum (L.). Journal of Parasitology, 82,203-209.

McSwain, J.L., Essenberg, R.C. & Sauer, J.R. (1982) Protein changes in the salivary glands of the lone star tick, Amblyomma americanum, during feeding. Journal of Parasitology, 68, 100-1 06.

Moorhouse, D.E. (1969) The attachment of some ixodid ticks to their natural host. Proceedings of the 2nd International Congress Acarology, Notringham, U.K., 1967, pp. 3 19-327. Akademia Kiado, Budapest.

Mustard, J.F. & Packham, M.A. (1977) Normal and abnormal haemostasis. British Medical Bulletin, 33, 187-192.

Neitz, A.W.H. & Vermeulen, N.M.J. (1987) Biochemical studies on the salivary glands and haemolymph of Amblyomma hebraeum. Onderstepoort Journal of Veterinary Research, 54,443-450.

Needham, G.R. & Teel, P.D. (1991) Off-host physiological ecology of ixodid ticks. Annual Review of Entomology, 36, 659-68 1.

1895- 1898.

48,779-782.

79-92.

208.532-54 I .


Tick saliva 285

Nuttall, G.H.F. & Strickland, C. (1908) On the presence of an anticoagulin in the salivary glands and intestines of Argas persicus. Parasitology,

Oaks, J.F., McSwain, J.L., Bantle, J.A., Essenberg, R.C. & Sauer, J.R. (1991) Putative new expression of genes in ixodid tick salivary gland development during feeding. Journal of Parasitology, 77,378-383.

Oliver, J.H., Owsley, M.R., Hutcheson, H.J., James, A.M., Chen, C., Irby, W.S., Dotson, E.M. & McClain, D.K. (1993) Conspecificity of the ticks Ixodes scapularis and I.dammini (Acari: Ixodidae): Journal of Medical Entomology, 30,54-63.

Packham, M.A. (1994) Role of platelets in thrombosis and hemostasis. Canadian Journal of Pharmacology, 72,278-284.

Radomski, M.W., Palmer, R.M.J. & Moncada, S. (1987) The anti- aggregating properties of vascular endothelium: interactions between prostacyclin and nitric oxide. British Journal of Pharmacology, 92,

Ramachandra, R.N. & Wikel, S.K. (1992) Modulation of host-immune responses by ticks (Acari: Ixodidae): Effect of salivary gland extracts on host macrophages and lymphocyte cytokine production. Journal of Medical Entomology, 29,8 18-826.

Ramachandra, R.N. & Wikel, S.K. (1995) Effects of Dermacentorandersoni (Acari: Ixodidiae) salivary gland extracts on Bos indicus and B.taurus lymphocytes and macrophages: in v i m cytokine elaboration and lymphocyte blatogenesis. Journal of Medical Entomology, 33,338-345.

Riek, R.F. (1962) Studies on the reaction of animals to infestation with ticks. VI. Resistance of cattle to infestation with the tick Boophilus microplus (Canestrini). Ausrralian Journal of Agriculrural Research,

Ribeiro, J.M.C. (1987) Role of saliva in blood-feeding by arthropods. Annual Review of Entomology, 32,463-478.

Ribeiro, J.M.C., Makoul, G.T., Levine, J., Robinson, D.R. & Spielman, A. (1985) Antihemostatic, antiinflammatory and immunosuppressive properties of the saliva of a tick, Ixodes dammini. Journal of Experimental Medicine, 161,332-344.

Ribeiro, J.M.C. & Spielman, A. (1986) Ixodes dammini: salivary anaphylatoxin inactivating activity. Experimental Parasitology, 62,

Ribeiro, J.M.C., Mather, T.N., Piesman, J. & Spielman, A. (1987) Dissemination and salivary delivery of Lyme disease spirochetes in vector ticks (Acari: Ixodidae). Journal of Medical Entomology, 24,

Ribeiro, J.M.C., Makoul, G.T. & Robinson, D.R. (1988) Ixodesdammini; evidence for salivary prostacyclin secretion. Journal of Parasitology,

Ribeiro, J.M.C., Weis, J.J. &Telford, S.R. (1990) Saliva of the tick Ixodes dammini inhibits neutrophil function. Experimental Parasitology, 70,

Ribeiro, J.M.C., Endris, T.M. & Endris, R. (1991) Saliva of the soft tick, Ornirhodorus moubata, contains anti-platelet and apyrase activities. Comparative Biochemistry and Physiology, 100, 109-1 12.

Ribeiro, J.M.C., Evans, P.M., McSwain, J.L. & Sauer, J.R. (1992) Amblyomma americanum: Characterization of salivary prostaglandins E, and F2- by RP-HpLChioassay and gas chromatography-mass spectrometry. Experimental Parasitology, 74, 1 12-1 16.

Ross, 1.C. (1926) An experimental study of tick paralysis in Australia. Parasitology, 18,410429.

Sabbatani, L. (1898) Ferment0 anticoagulante dell’ Ixodes ricinus. Giornale della Accademia di Reale Medicina di Torino, 4S.IV, 380-395.

Sauer, J.R. & Hair, J.A. (1972) The quantity of blood ingested by the lone star tick (Acarina: Ixodidae). Annals of the Entomological Society ofAmerica, 65, 1065-1068.

Sauer. J.R., Bowman, A.S., McSwain, J.L. & Essenberg, R.C. (1996) Salivary gland physiology of blood-feeding arthropods. In: The Immunology of Host-Ectoparasific Arrhropod Relationships (ed. by

1,302-310.

639-646.

13,532-550.

292-297.

20 1-205.

74, 1068-1 069.

382-388.

S. K. Wikel). pp. 62-84. CAB International, Wallingford. Sauer, J.R., McSwain, J.L., Bowman, A S . & Essenberg, R.C. (1995)

Tick salivary gland physiology. Annual Review of Entomology, 40,

Silldorff, E.P.,Yang, S. & Pallone, T.L. (1995) Prostaglandin E,abrogates endothelin-induced vasoconstriction in renal outer medullary descending vasa recta of therat. Jourticil uf Clinical Investigoriv11,95,2734-2740.

Soneshine, D.E. ( I 991) Biology of Ticks, Vol. I . Oxford University Press. Stark, K.R. & James, A.A. (1995) A factor Xa-directed anticoagulant

from the salivary glands of the yellow fever mosquito Aedes aegypri. Experimental Parasitology. 81,321-33 I .

Stark, K.R. & James, A.A. (1996a) Salivary gland anticoagulants in Culicine and Anopheline mosquitoes (Diptera: Culicidae). Journal of Medical Entomology, 33,645-650.

Stark, K.R. & James, A.A. (1996b) Anticoagulants in vector arthropods. Parasitology Today, 12,430437.

Stitch, R.W., Sauer, J.R., Bantle, J.A. & Kocan, K.M. (1993) Detection of Anaplasma marginale (Rickettsiales: Anaplasmataceae) in secretagogue-induced oral secretions of Dermacentor andersoni (Acari: Ixodidae) with the polymerase chain reaction. Journal of Medical Entomology, 30,789-794.

Trager, W. (1939) Acquired immunity to ticks. Journal of Parasitology,

Urioste, S., Hall, L.R., Telford, S.R., I11 &Titus, R.G. (1994) Saliva of the Lyme disease vector, Ixodes dammini, blocks cell activation by a non-prostaglandin E,-dependent mechanism. Journal of Experimental Medicine, 180, 1077-1085.

Wang, H. & Nuttall, P.A. (1994) Excretion of host immunoglobulin in tick saliva and detection of IgG-binding proteins in tick haemolymph and salivary glands. Parasitology, 109,525-530.

Wang, H. & Nuttall, P.A. (l995a) Immunoglobulin-G binding proteins in the ixodid ticks, Rhipicephalus appendicularus, Amblyomma variegatum and Ixodes hexagonus. Parasirology, 111, 161-165.

Wang, H. & Nuttall, P.A. (l995b) Immunoglobulin G binding proteins in male Rhipicephalus appendiculatus ticks. Parasire Immunology, 17,

Wang, X., Coons, L.B., Taylor, D.B., Stevens, S.E., Jr & Gartner, T.K. (1996) Variabilin, a novel RGD-containing antagonist of GPIIb-Illa and platelet aggregation inhibitor from the hard tick Dermacentor variabilis. Journal of Biological Chemistry, 271, 17785- 17790.

Waxman, L., Smith, D.E., Arcuri, K.E. & Vlasuk, G.P. (1990) Tick anticoagulant peptide (TAP) is a novel inhibitor of blood coagulation factor Xa. Science, 248,593-596.

Waxman, L. & Connolly, T.M. (1993) Isolation of an inhibitor selective for collagen-stimulated platelet aggregation from the soft tick Ornithodoros moubata. Journal of Biological Chemistry, 268,5445- 5449.

Wikel, S.K. (1996) Host immunity to ticks. AnnualReview ofEntornology, 41, 1-22.

Wikel, S.K.. Ramachandra, R.N. & Bergman, D.K. (1994) Tick-induced modulation of the host immune response. International Journal for Parasitology, 24,59-66.

Zeidner, N., Dreitz, M., Belasco, W. & Fish, D. (1996) Suppression of acute Ixodes scapularis-induced Borrelia burgdorferi infection using tumor necrosis factor-a, interleukin-2 and interferon-y. Journal of Infectious Diseases, 173, 187-195.

Zhu, K., Sauer, J.R., Bowman,A.S. & Dillwith, J.W. (1997a) Identification and characterization of anticoagulant activity in the lone star tick, Amblyomma americanum (L.). Journal of Parasitology, 8 3 , 3 8 4 3 .

Zhu. K., Dillwith, J.W., Bowman, A S . & Sauer, J.R. (1997b) Identification of hemolytic activity in the saliva of the lone star tick (Acari: Ixodidae). Journal of Medical Entomology,M, 160-1 66.

245-267.

25.57-8 I .

5 17-524.

Accepted 6 February I997

0 1997 Blackwell Science Ltd, Medical and Veferinary Entomology 11: 277-285

Tick saliva: recent advances and implications for vector competence

Documents

Transcript of Tick saliva: recent advances and implications for vector competence