Following probing and injury of blood vessels by tick mouthparts, arachidonic acid is released by activated platelets and is converted into thromboxane A₂, a platelet-aggregating, platelet-degranulating, and vasoconstricting substance. Activated platelets release serotonin which, together with thromboxane A₂, is responsible for early vasoconstriction in local inflammation caused by tissue injury. To antagonize vasoconstrictors produced by the host at the site of tissue injury, vasodilators are secreted by ticks to the feeding pool. To date, only non-proteinaceous vasodilatory compounds have been identified in tick saliva. These include lipid derivatives such as prostacyclin and prostaglandins (Ribeiro et al., 1988, 1992; Bowman et al., 1996). However, a tick histamine release factor (tHRF), secreted in Ixodes scapularis saliva (Dai et al., 2010) and a novel Ixodes ricinus serine proteinase inhibitor (serpin) named IRS-2, which inhibits cathepsin G and chymase (Chmelar et al., 2011), probably also act as modulators of vascular permeability (Chmelar et al., 2012).

Platelet aggregation represents the initial and most immediate stage of haemostasis. Following vascular injury, platelets adhere to the subendothelial tissue and become activated by agonists such as collagen, thrombin, adenosine diphosphate (ADP), and thromboxane A₂. Agonists bind to specific receptors on the surface of platelets and initiate a long and highly complex chain of intracellular chemical reactions that lead to platelet aggregation and the formation of a heamostatic plug. The platelet aggregation cascade is targeted by ticks at several stages (Francischetti, 2010). A strategy used by a number of ticks is targeting ADP, an agonist important for completion of platelet aggregation, via salivary apyrase. Apyrase, an adenosine triphosphate (ATP)-diphosphohydrolase enzyme, hydrolyses the phosphodiester bonds of ATP and ADP. Apyrase activity has been demonstrated in the salivary glands and saliva of both soft ticks (Ribeiro et al., 1991; Mans et al., 1998a,b, 2002a) and hard ticks (e.g., I. scapularis; Ribeiro et al., 1985). Apyrase from Rhipicephalus (Boophilus) microplus belongs to the 5′-nucleotidase family (Liyou et al., 1999). On the other hand, apyrase activity has not been detected in the saliva of, e.g., Amblyomma americanum (Ribeiro et al., 1992), but increased prostaglandin levels in the saliva of this tick inhibit platelet aggregation by preventing ADP secretion during platelet activation (Ribeiro et al., 1992; Bowman et al., 1995).

Some of the tick-derived platelet aggregation inhibitors interfere with the interaction of collagen with platelet receptors. Activation of platelets by collagen is prevented, e.g., by Moubatin, a specific inhibitor of collagen stimulated platelet activation from Ornithodoros moubata, while tick adhesion inhibitor (TAI) identified in the same tick species inhibits the adhesion of platelets to matrix collagen (Waxman and Connolly, 1993; Karczewski et al., 1995). Moubatin belongs to the family of lipocalins and probably prevents platelet aggregation caused by ADP released from collagen-activated platelets (Valenzuela, 2004). Longicornin, another inhibitor of collagen-mediated platelet aggregation, was isolated from the hard tick Haemapysalis longicornis (Cheng et al., 1999). However, Longicornin does not bind directly to collagen fibers and does not affect platelet adhesion to collagen, indicating that the inhibitor, similarly to Moubatin, shares a common receptor with collagen.

Thrombin is a key enzyme in thrombosis and haemostasis. In addition to its main role in the formation of the fibrin clot, it induces platelet aggregation. Three functional sites have been recognized in thrombin—the active site, the anion-binding exosite I that mediates binding of thrombin to fibrinogen, the platelet receptor and thrombomodulin, and the anion-binding exosite II, which is the heparin-binding site. Salivary antithrombins detected in both soft ticks and hard ticks that are involved in the inhibition of the coagulation cascade, also inhibit thrombin-induced platelet aggregation (Hoffmann et al., 1991; Nienaber et al., 1999; Kazimírová et al., 2002). The serpin IRS-2 from I. ricinus inhibits both cathepsin G- and thrombin-induced platelet aggregation (Chmelar et al., 2011).

Post-activation inhibitors of platelet aggregation target the platelet fibrinogen receptor. Activated platelets express surface adhesion receptor proteins, known as integrins that enable cell-cell and cell-matrix interactions. As the platelets are activated by platelet agonists (thrombin, collagen, and ADP), the ligands [fibronectin, vitronectin, von Willebrand factor, which have the common Arg—Gly—Asp (RGD) sequence, and fibrinogen] bind to glycoprotein (GP)IIb-IIIa via their RGD motif. Interaction between the fibrinogen and the GPIIb-IIIa complex is the important final step in platelet aggregation. Non-RGD disintegrins block the binding of fibrinogen to the integrin αIIbβ3, which is a fibrinogen receptor on surface of activated platelets. The αIIbβ3 antagonists can displace fibrinogen from its receptor thereby allowing disaggregation. Tick-derived disintegrin-like peptides such as Savignygrin (Mans et al., 2002b) and Variabilin (Wang et al., 1996) contain the integrin recognition motif RGD used for binding to GPIIb-IIIa and can inhibit platelet aggregation by preventing the binding of other ligands to the platelet receptor. In contrast, Disaggregin, a fibrinogen receptor antagonist from the soft tick O. moubata, is a GPIIb-IIIa antagonist, which lacks the RGD motif and inhibits platelet aggregation by preventing binding to ligands by distinct mechanisms from disintegrin-like peptides (Karczewski et al., 1994). Ixodegrins from Ixodes pacificus and I. scapularis display sequence similarity to Variabilin, with two additional cysteines in the RGD position (Francischetti et al., 2005b), but their disintegrin activity has yet to be confirmed (Francischetti, 2010).

Disaggregation of platelet aggregates is considered an important back-up mechanism that ticks can use if first-line defence mechanisms fail to inhibit platelet aggregation (Mans and Neitz, 2004). Aggregated platelets may be disaggregated by the removal of fibrinogen from the fibrinogen receptor by competitive binding of an antagonist to the fibrinogen receptor. Proteolysis of fibrinogen can also lead to platelet disaggregation. Apyrase from the soft tick O. moubata displays a disaggregation effect on aggregated platelets (Mans et al., 2000), whereas the GPIIb-IIIa antagonist Savignygrin from O. savignyi can displace fibrinogen from its receptor and lead to disaggregation (Mans et al., 2002c).

Blood coagulation involves a series of enzymatic reactions where an inactive proenzyme (coagulation factor) is converted to an active form, which then activates the next proenzyme in the cascade. Thrombin is involved in the final common pathway of the coagulation cascade and converts fibrinogen into fibrin clots, but also regulates the activity of blood coagulation factors and stimulates platelet reactions. Ticks have evolved powerful tools to prevent or prolong blood coagulation throughout their extended blood meal. A number of inhibitors of serine proteinases that are involved in the coagulation cascade have been identified and characterized from ticks. The majority of inhibitors identified so far are proteins that display a variety of molecular masses, targets and inhibitory mechanisms (Koh and Kini, 2009; Kazimírová et al., 2010). Based on the mechanism of action, anticoagulants from ticks can be classified as: thrombin inhibitors; inhibitors of activated factor X (FXa); inhibitors of the extrinsic tenase complex (ETC); contact system protein inhibitors (Koh and Kini, 2009), with thrombin and FXa being the most common targets.

Innate immune responses represent the first line of immune defence of the hosts to local injury and involve complement, acute phase proteins, neutrophils, macrophages, mast cells, basophils, eosinophils, dendritic cells (DCs) and NK cells. Complement components, prostaglandins, leukotrienes, chemokines, and cytokines contribute to the recruitment of inflammatory cells to the site of injury (e.g., Andrade et al., 2005). Normally, the consequences of prolonged feeding of an ectoparasite would be local inflammation and rejection. However, ticks produce compounds that inhibit or modulate the pro-inflammatory functions of most cell types infiltrating the attachment site, e.g., neutrophils (Ribeiro et al., 1990; Guo et al., 2009), NK cells (Kubeš et al., 1994), macrophages (Kopecký and Kuthejlová, 1998; Kramer et al., 2011), T cells (e.g., Ramachandra and Wikel, 1992; Bergman et al., 2000) and DCs (Cavassani et al., 2005; Skallová et al., 2008).

The skin is the main organ of the tick-host interface, playing a crucial role in the response of the host to tick infestation as well as in pathogen transmission by the vector. Local modulation of cutaneous immune responses at the tick bite site occurs soon after tick attachment and is characterized by modulation of responses in resident cells that merge into a neutrophil-driven immune response a few hours post-attachment (Heinze et al., 2012a). Ir-LBP, a lipocalin present in I. ricinus was shown to inhibit neutrophil chemotaxis in vitro and host inflammatory response in vivo by decreasing the numbers and activation of neutrophils located at the tick bite site, impairing neutrophil function in inflammation (Beaufays et al., 2008). It was also demonstrated that due to an antialarmin effect on human primary keratinocytes, saliva of I. ricinus inhibits cutaneous innate immunity and migration of immune cells to the tick bite site, threreby creating favorable conditions for tick-borne pathogens that are transmitted to and multiply in the host skin (Marchal et al., 2009, 2011).

Recruitment of specific leukocyte populations during the inflammatory response is triggered by chemokines that are key mediators of the inflammatory response against parasites. Ticks have evolved various strategies to manipulate the host cytokine network. The chemokine CXCL8 [interleukin(IL)-8] is a chemo-attractant for neutrophils. Anti-IL-8 activity impairing neutrophil functions was reported from the saliva of various hard ticks (Hajnická et al., 2001). Moreover, tick saliva contains a variety of inhibitory activities directed against other pro-inflammatory cytokines such as IL-2 and the chemokines CCL2 (MCP-1), CCL3 (MIP-1α), CCL5 (RANTES), and CCL11 (eotaxin) (Hajnická et al., 2005). These activities are tick species-, developmental stage-, sex- and feeding stage-specific (Vančová et al., 2010b), but the anti-cytokine factors have not been identified. In contrast, Evasins, a family of novel chemokine binding proteins have been detected in salivary glands of R. sanguineus ticks (Frauenschuh et al., 2007). Evasins show selectivity to different chemokines: Evasin-1 binds to CCL3, CCL4, and CCL18; Evasin-3 binds to CXCL8 and CXCL1; and Evasin-4 binds to CCL5 and CCL11 (Frauenschuh et al., 2007; Déruaz et al., 2008). Evasin-3-like activities were also demonstrated for other metastriate tick species, providing further evidence that ticks control host neutrophil functions during feeding (Vančová et al., 2010a). Hyalomma asiaticum asiaticum ticks evade host immune reactions by modulating cytokine secretion and detoxification of free radicals (Wu et al., 2010). Two families of immunoregulatory peptides, Hyalomin-A and -B, identified in salivary glands of this species, suppress host inflammatory responses either by inhibiting secretion of tumor necrosis factor (TNF)-alpha, monocyte chemotactic protein-1 (MCP-1), and interferon (IFN)-gamma or by increasing the secretion of the immunosuppressant cytokine IL-10.

Modulation of wound healing and angiogenesis seems to be another strategy used by ticks to suppress host inflammatory responses and succeed in prolonged blood feeding (Francischetti et al., 2009; Hajnická et al., 2011). Tick salivary compounds have been shown to bind the transforming growth factor (TGF)-β1, the platelet-derived growth factor (PDGF), the fibroblast growth factor (FGF)-2 and the hepatocyte growth factor (HGF) in a species-specific manner (Hajnická et al., 2011). Dermacentor variabilis saliva suppresses basal and PDGF-stimulated fibroblast migration and reduces extracellular signal-regulated kinase (ERK) activity stimulated with PDGF, suggesting that ticks ensure prolonged maintenance of the feeding lesion in the host skin also by the suppression of ERK activation and fibroblast migration, i.e., important events in wound healing (Kramer et al., 2008).

In addition to growth-factor binding capacities, distinct tick salivary molecules with similarities to disintegrin metalloproteases and thrombospondin are involved in cell-matrix interactions and/or the inhibition of angiogenesis (Valenzuela et al., 2002; Francischetti et al., 2005a; Fukumoto et al., 2006; Harnnoi et al., 2007). The proteins ISL 929 and ISL 1373 with homology to the cysteine-rich domain of disintegrin metalloproteinases which were derived from the sialome of I. scapularis, reduce the expression of β2 integrins and impair the adherence of polymorphonuclear leukocytes (PMNs) (Guo et al., 2009). Inhibition of microvascular endothelial cell proliferation by the saliva of I. scapularis (Francischetti et al., 2005a) suggests that a metalloprotease is responsible for this activity. In addition to its anticoagulation properties, the Kunitz-like serine proteinase inhibitor Ixolaris from I. scapularis downregulates the vascular endothelial growth factor and reduces vessel density in tumours (Carneiro-Lobo et al., 2009). A troponin I-like molecule (HLTnI) present in varius organs, including salivary glands of H. longicornis, also inhibits capillary formation of human vascular endothelial cells (Fukumoto et al., 2006) and Haemangin, a Kunitz-type protein from the salivary glands of the same species, displays similar effects on angiogenesis and wound healing (Islam et al., 2009). These results indicate that tick anti-angiogenic factors, in addition to their inhibitory effects on angiogenesis, may also play an important role in controlling tick attachment and pathogen transmission.

Bradykinin and histamine are important mediators of itching and pain and could stimulate host grooming and removal of the feeding ticks. However, ticks developed efficient countermeasures to these host reactions. Tick salivary kininases have been shown to hydrolyse circulating kinins (e.g., bradykinin). For example, a dipeptidyl carboxypeptidase activity was found to account for the kininase activity of I. scapularis saliva (Ribeiro and Mather, 1998). In addition, amine-binding proteins of the lipocalin family that suppress host responses to local inflammation are produced by hard ticks. A male-specific histamine-binding salivary protein [RaHBP(M)] and two female-specific histamine-binding salivary proteins [RaHBP(F)-1,2] were isolated from the saliva of R. appendiculatus (Ra) (Paesen et al., 1999) and the gene for a protein that binds both serotonin and histamine (SHBP) was identified in Dermacentor reticulatus (Sangamnatdej et al., 2002). Recently, a tick derived protease inhibitor (TdPI) has been described and characterized from R. appendiculatus (Paesen et al., 2007). TdPI suppresses the activity of human β-tryptases, i.e., mast cell-specific serine proteases with roles in inflammation and tissue remodeling.

Another category of compounds produced by ticks to evade host immune responses are proteins that mimic host proteins. Tick macrophage migration inhibitory factor (MIF) is a peptide detected in salivary glands of the hard tick Amblyomma americanum (Jaworski et al., 2001). This peptide inhibits the migration of macrophages and probably protects the feeding ticks from macrophage attack.

Non-proteinaceous substances, like purine nucleoside adenosine (Ado) and prostaglandin PGE₂ present in saliva of R. sanguineus, are also involved in the modulation of host inflammatory and immune responses. These compounds inhibit the production of pro-inflammatory IL-12p40 and TNF-alpha and stimulate the production of anti-inflammatory IL-10 by murine DCs (Oliveira et al., 2011).

The complement system links the innate and adaptive responses of the host immune system and is activated via three main pathways (alternative, classical, and lectin pathway), whereby the alternative pathway is the major line of defence against invading pathogens and is also involved in resistance to ticks. SGE of ixodid ticks were found to inhibit complement activity of vertebrates, whereby the anti-complement activities correlated to the reported host range of the tested tick species (Lawrie et al., 1999). Subsequently, several molecules with anti-complement activities were identified in tick salivary glands. Isac, Salp 20 and Isac-1 from I. scapularis (Valenzuela et al., 2000; Tyson et al., 2007) and the Isac paralogues IRAC I and II from I. ricinus (Daix et al., 2007; Couvreur et al., 2008) specifically inhibit formation of the C3 convertase of the alternative pathway by blocking binding of complement factor B to complement C3b. On the other hand, OmCI (Ornithodoros moubata complement inhibitor) belonging to proteins of the lipocalin family has been the first natural complement inhibitor isolated from a soft tick that specifically targets the C5 activation step in the complement cascade (Nunn et al., 2005).

During the first exposure to ticks, immunoglobulin and T cell-mediated immune responses are induced in the hosts. Salivary immunogens are processed by Langerhans cells located in the epidermis and presented to immunocompetent lymphocytes (Schoeler and Wikel, 2001; Andrade et al., 2005). Antigen presenting cells can also transport immunogens to draining lymph nodes and promote antibody- and cell-mediated responses. Delayed type hypersensitivity response characteristic of influx of lymphocytes and macrophages, basophils and eosinophils is often observed at the tick feeding site. Homocytotropic antibodies are produced and memory B and T lymphocytes are generated.

Immune resistance to ticks is important in protection from infestation with these ectoparasites and consequently also contributes to the reduction in pathogen transmission from infected ticks to the hosts (Wikel et al., 1997), although specific antigenic and functional components of tick saliva have not been well characterized. In resistant hosts (e.g., rabbits, guinea pigs), the presence of reactive antibodies and effector T lymphocytes assures a rapid response to infestation and can impair tick feeding, whereas ticks have evolved to overcome host immune responses in natural tick-host associations (Ribeiro, 1995). I. scapularis salivary antigens that elicit antibodies in resistant hosts have been determined based on screening of salivary gland cDNA expression library with tick-immune mice sera. Using this procedure, the presence of Salp25D, a protein which neutralizes the effect of reactive oxygen species produced by activated neutrophils, has been detected (Das et al., 2001; Narasimhan et al., 2007b). In contrast to resistant hosts, mice generally do not develop acquired resistance to repeated tick feeding (e.g., Schoeler et al., 1999); however, during secondary tick infestation, their cytokine response displays a mixed Th1/Th2 profile and enhanced activity of regulatory T cells (Heinze et al., 2012b).

A variety of tick species have been found to suppress the in vitro proliferation of lymphocytes induced with T and/or B cell mitogens. Tick-induced immunosuppression of the host is also characterized by decreased primary antibody responses to T cell-dependent antigens. Moreover, ticks have evolved ways to alter the production of T lymphocyte cytokines. Generally, it has been reported that tick saliva polarizes the host immune response toward a Th2 type profile characterized by the down-regulation of pro-inflammatory Th1 cytokines (IL-2, IFN-gamma) and enhanced production of Th2 cytokines (IL-4, IL-5, IL-6, IL-10, IL-13) (see Gillespie et al., 2000; Schoeler and Wikel, 2001; Wikel and Alarcon-Chaidez, 2001; Brossard and Wikel, 2008, and references therein). It has been suggested that inhibition of T cell responsiveness to mitogens could result from the direct effect of salivary gland proteins on lymphocytes or from their production of IL-10, while up-regulation of IL-4 and IL-10 probably leads to the development of a Th2 response (Ramachandra and Wikel, 1992; Wikel, 1999; Schoeler and Wikel, 2001; Wikel and Alarcon-Chaidez, 2001).

Several T cell inhibitors have been identified in ticks. A 36 kDa protein (P36) suppressing T cell proliferation is present in the saliva of feeding D. andersoni (Bergman et al., 2000). Iris was detected in the salivary glands of I. ricinus females (Leboulle et al., 2002). The production of Iris is induced in the tick salivary glands during the feeding process and the protein is secreted into the tick saliva. It suppresses T cell proliferation, induces a Th2 type immune response and inhibits the production of pro-inflammatory cytokines IL-6 and TNF-alpha. Salp15, a 15 kDa salivary gland protein from I. scapularis is another feeding-induced protein that inhibits the activation of T cells. Salp15 specifically binds to the CD4 molecules on the surface of CD4+ T (helper) cells, which results in inhibition of T cell receptor-mediated signaling, leading to reduced IL-2 production and impaired T cell proliferation (Anguita et al., 2002; Garg et al., 2006). In addition, Salp15 impairs DCs functions by inhibiting Toll-like receptor- and Borrelia burgdorferi-induced production of pro-inflammatory cytokines by DCs and DC-induced T cell activation (Hovius et al., 2008a). Evidence was also provided that the pathogen B. burgdorferi in I. scapularis exploits Salp15 during transmission to a vertebrate host, as it specifically interacts with B. burgdorferi outer surface protein C (OspC) and the binding of Salp15 protects B. burgdorferi from antibody-mediated killing in vitro (Ramamoorthi et al., 2005). Salp 15-like sequences encoding proteins of the Salp family have also been identified in salivary glands of Ixodes pacificus, I. ricinus, and I. persulcatus, which are other major vectors of disease agents in the USA and Eurasia (Hovius et al., 2007; Hojgaard et al., 2009; Mori et al., 2010). The results suggest that the Salp15 homologues can be involved in host immunomodulation and transmission of Borrelia species in the above regions.

Other immunomodulatory proteins facilitating tick feeding and pathogen transmission were also detected in the saliva of I. scapularis: a secreted IL-2 binding protein that suppresses T cell proliferation and the activity of immune effector cells responsive to IL-2 stimulation (Gillespie et al., 2001), and the salivary cysteine protease inhibitors sialostatin L and sialostatin L2, with inhibitory activity against cathepsin L (Kotsyfakis et al., 2006). Sialostatin L displays anti-inflammatory properties and inhibits proliferation of cytotoxic T lymphocytes and LPS-induced maturation of DCs, whereas sialostatin L2 does not modulate functions of antigen presenting cells, but is probably important for successful tick feeding (Kotsyfakis et al., 2006; Sá-Nunes et al., 2009). In addition, sialostatin L2 stimulates the growth of B. burgdorferi in murine skin, however, the mechanism of this growth stimulation has not been revealed (Kotsyfakis et al., 2010).

Ticks can also benefit from the suppression of B cell responses of vertebrate hosts by inhibiting the production of specific anti-tick antibody responses that could cause rejection of feeding ticks by the host. B cell inhibitory proteins (BIP and BIF) have been identified in I. ricinus and H. asiaticum asiaticum, respectively (Hannier et al., 2004; Yu et al., 2006). Along with feeding ticks, tick-borne pathogens like B. burgdorferi might also benefit from BIP-mediated B cell suppression in their vertebrate hosts (Hannier et al., 2004).

In addition to substances modulating the host immune responses, ticks produce immunoglobulin (IgG)-binding proteins that bind ingested host IgGs and excrete them by salivation. This mechanism protects the ticks primarily from ingested host immunoglobulin's and facilitates their feeding (Wang and Nuttall, 1999).

A novel mechanism of tick-induced modulation of host adaptive immunity which may facilitate pathogen transmission has been discovered recently (Preston et al., 2013). Japanin, a salivary gland protein from R. appendiculatus belonging to a new clade of lipocalins from metastriate ticks, was found to target DCs. Japanin specifically reprogramms responses of DCs to a wide variety of stimuli in vitro, altering their expression of co-stimulatory and co-inhibitory transmembrane molecules and secretion of pro-inflammatory, anti-inflammatory and T cell polarizing cytokines and it also inhibits the differentiation of DCs from monocytes.

The phenomenon of SAT was first described for the Thogoto virus (THOV)—R. appendiculatus association. Increased THOV transmission to uninfected R. appendiculatus nymphs was observed when the nymphs fed on animals inoculated with a mixture of the virus and SGE of tick females compared to nymphs feeding on animals inoculated with virus only (Jones et al., 1989). Enhanced infection of ticks feeding on animals experimentally inoculated with pathogens and tick saliva (or SGE), i.e., direct evidence of SAT (Nuttall and Labuda, 2008), has subsequently been reported for a few other pathogens, e.g., tick-borne encephalitis virus (TBEV) (Labuda et al., 1993a), B. burgdorferi s.l. (Pechová et al., 2002; Zeidner et al., 2002; Macháčková et al., 2006; Horká et al., 2009) and Francisella tularensis (Kročová et al., 2003) (see Table 2).

In studies involving Borrelia spirochaetes, injection of borreliae together with I. ricinus or I. scapularis SGE increased the level of bacteraemia in the murine host, enhanced the transmission of spirochaetes to feeding ticks and suppressed the production of pro-inflammatory cytokines in draining lymph nodes of mice (Pechová et al., 2002; Zeidner et al., 2002). Moreover, SGE of I. ricinus inhibited killing of B. garinii by murine macrophages and reduced the production of two major defence molecules of phagocytosis—superoxide and nitric oxide (Kuthejlová et al., 2001). Saliva of I. scapularis reduced PMN adhesion via downregulation of beta2-integrins and decreased the efficiency of PMN in the uptake and killing of spirochaetes, thus facilitating the transmission and initial survival of spirochaetes (Montgomery et al., 2004). The SAT compounds responsible for the described effects have not been identified, but they probably depend on vector competence of individual tick species for the pathogen and can vary with different pathogens (Nuttall and Labuda, 2008).

Studies on non-viraemic transmission (NVT) of pathogens from infected to non-infected ticks co-feeding on the same host provide indirect evidence of SAT (Nuttall and Labuda, 2008). By mimicking the natural conditions when infected and non-infected ticks feed in aggregates on a vertebrate host, Jones et al., 1987 demonstrated that transmission of THOV from infected to non-uninfected R. appendiculatus ticks co-feeding on non-viraemic guinea pigs was more efficient than transmission on higly viraemic hamsters, and suggested a novel mode of arthropod-borne virus transmission. NVT, independent of a systemic infection of a host, has subsequently been shown for other tick-pathogen associations, mainly TBEV and other arthropod-borne viruses (see Nuttall and Labuda, 2008). Because NVT of TBEV occurs on both susceptible and non-susceptible hosts and can also occur in the presence of virus-specific neutralizing antibodies, it is considered one of the main mechanisms of the maintenance of TBEV in natural foci (Labuda et al., 1993a; Randolph et al., 1999; Randolph, 2011).

Immunomodulation of the tick attachment site by tick salivary compounds is suggested to play a crucial role in the process of NVT as the local skin site of tick attachment is an important focus of viral replication early after transmission (Labuda et al., 1996). It was clearly shown that transmission of TBEV from infected to non-infected I. ricinus ticks feeding together on mice was correlated with infection in the skin. The virus was recruited in migratory Langerhans cells and neutrophils preferentially to the site in which ticks were feeding compared with uninfested skin sites and migratory monocyte/macrophages produced infectious virus.

Although the maintenance of the B. burgdorferi s.l. spirochaetes depends largely on systemic transmission, transmission of the bacteria between co-feeding ticks has also been demonstrated (Gern and Rais, 1996; Ogden et al., 1997). In laboratory models, duration of infectivity, density and distance between co-feeding ticks have been established as important factors affecting efficiency of transmission of the spirochaetes (Piesman and Happ, 2001; Richter et al., 2002). However, such models seldom mimic the situation in nature. On the other hand, field studies revealed that although sheep do not support systemic infections of B. burgdorferi, in the absence of alternative hosts, they can transmit localized infections from infected to uninfected ticks co-feeding at the same skin site (Ogden et al., 1997). Due to the presence of various Borrelia genospecies and their associations to certain groups of reservoir hosts, the extent and importance of non-systemic transmission in the ecology of Lyme borreliosis needs to be further explored (Randolph et al., 1996; Ogden et al., 1997; Randolph, 2011).

Immunomodulatory capacities of tick saliva are considered key factors in pathogen transmission. It has been demonstrated that the local skin site infested with ticks and modulated by tick saliva is an important focus of virus replication early after TBEV transmission by ticks. Cellular infiltration and cell migration at the tick attachment site may also facilitate pathogen transmission between infected and uninfected co-feeding ticks (Labuda et al., 1996). These findings were supported by a study where the effects of TBEV infection on DCs and their modulation by I. ricinus saliva were demonstrated in vitro, showing that treatment of the cells with tick saliva increased the proportion of virus-infected cells and decreased the virus-induced production of TNF-alpha and IL-6 and reduced virus-induced apoptosis (Fialová et al., 2010).

There is increasing evidence that the host immune reactions to B. burgdorferi and consequently the outcome of Borrelia infection in the host and its infectivity for ticks depend on the presence of the vector. It was demonstrated that mice infected with B. burgdorferi via I. ricinus were more infective for subsequently attached ticks than those experimentally inoculated with the spirochaete (Gern et al., 1993). BALB/c mice developed a Th2 immune response against B. burgdorferi after tick inoculation and a mixed Th1/Th2 response after syringe inoculation. Moreover, in comparison with syringe inoculation of B. burgdorferi, IL-4 produced in host draining lymph nodes following tick bites greatly inhibited the production of anti-borrelial IgG2a antibodies (Christe et al., 2000). These findings were further supported by experiments in which a B cell inhibitory protein (BIP) from I. ricinus salivary glands suppressed B lymphocyte proliferation induced by the B. burgdorferi OspC, suggesting that BIP may play an important role in enhancing B. burgdorferi transmission by the tick (Hannier et al., 2003).

Immunomodulation by tick saliva resulting in down-regulation of cytokines, chemokines and antimicrobial peptides in vertebrate hosts was shown to facilitate transmission and infection by Borrelia. The significance of the anti-inflammatory properties of I. ricinus SGE was demonstrated experimentally in transmission of B. garinii to mice when the bacteria were used to stimulate inflammation (Severinová et al., 2005). In these experiments, tick saliva injected together with spirochaetes reduced the numbers of leukocytes and T lymphocytes in the infected murine epidermis at early time-points post infection and decreased the total cell count in draining lymph nodes. Maturation of DCs (Skallová et al., 2008) as well as interactions of B. garinii and murine DCs were also impaired by I. ricinus saliva through the inhibition of proliferation and IL-2 production by specific CD4+ T cells and decreased production of Th1 and Th2 cytokines by DCs (Slámová et al., 2011). In addition, I. ricinus saliva modulated IFN-gamma signaling pathways in DCs (Lieskovská and Kopecký, 2012a) and pathways activated by Toll-like receptor (TLR-2) ligand in Borrelia-stimulated DCs (Lieskovská and Kopecký, 2012b).

The involvement of tick salivary compounds in the modulation of skin innate immunity mediated by antimicrobial peptides of the cathelicidin and defensin families in the course of Borrelia infection was also demonstrated. When spirochaetes were inoculated to mice, Borrelia triggered skin inflammation with induction of the cathelin-related antimicrobial peptide, the mouse cathelicidin and TNF-alpha. However, after natural transmission of the spirochaetes via feeding I. ricinus, the inflammatory genes were supressed, suggesting that saliva of the vector tick facilitate Borrelia establishment in the host skin (Kern et al., 2011).

Generally, repeated infestation of mice with pathogen-free Ixodes ticks results in a polarization of the host immune response toward the Th2 anti-inflammatory cytokine pattern, with a corresponding down-regulation of Th1 responses (Schoeler et al., 1999, 2000b; Mejri et al., 2001). Consequently, down-regulation of pro-inflammatory factors promotes the initial establishment and dissemination of spirochetal infection, but reconstitution of cytokines down-regulated by tick infestation provides protection against tick-transmitted B. burgdorferi (Zeidner et al., 1996, 1997). This was demonstrated in vivo when mice or Guinea pigs repeatedly infested with pathogen-free I. scapularis nymphs were protected against infection with B. burgdorferi transmitted via infected ticks, suggesting that immunity against the tick interferes with transmission of the spirochatete (Wikel et al., 1997; Nazario et al., 1998). Moreover, immunization of Guinea pigs with I. scapularis salivary gland proteins produced within the first day of tick attachment impaired B. burgdorferi transmission from ticks to hosts, probably by evoking acquired immunity against tick feeding (Narasimhan et al., 2007a).

Borrelia burgdorferi s.l. displays distinct phenotypic plasticity (Radolf et al., 2012) and exploits a number of tick proteins (Table 2) that support colonization and persistence of the pathogen in the vector and its transmission to the vertebrate host (Kung et al., 2013). Within an infected tick, spirochaetes express OspA and bind to the midgut wall of the tick by using a tick expressed protein (TROSPA) (Pal et al., 2004). After attachment of the tick to a host and onset of feeding, the spirochaetes start to express OspC and move from the midgut through the haemolymph to the salivary glands. In the tick salivary glands, spirochaetes bind to the secreted salivary protein, Salp15, which protects the spirochaetes from antibody-mediated killing and facilitates their transmission and replication in the host skin (Ramamoorthi et al., 2005). The spirochaetes are transmitted to the host with tick saliva containing various salivary molecules which modulate T cells (Salp15), complement (ISAC, Salp20), macrophages, neutrophils, and B cell activities (BIP) and other components of the host immune system (see above) and help Borrelia to infect and disseminate in the mammalian host.

Salp15 is the first tick SAT molecule and was identified in salivary glands of I. scapularis. Except for immunosuppressive functions, Salp15 is an immunoprotective antigen, because antiserum against the protein protects mice from Borrelia infection (Dai et al., 2009).

Salp15 Iric-1, a Salp15 homologue, was identified in I. ricinus, the vector of European Borrelia species. The protein was found to differentially protect B. burgdorferi s.s., B. garinii, and B. afzelii from antibody-mediated killing in the host (Hovius et al., 2008c).

Salp25D is another immunodominant salivary protein present in I. scapularis, which is important during tick acquisition of B. burgdorferi and acts as an antioxidant that facilitates pathogen survival (Das et al., 2001; Narasimhan et al., 2007b).

Salp20 protects B. burgdorferi from in vitro lysis and probably from components of the complement pathway during transmission from an infected tick to the host (Tyson et al., 2007).

A tHRF is up-regulated in I. scapularis salivary glands during the rapid feeding phase and probably facilitates tick engorgement and B. burgdorferi infection by modulation of vascular permeability and increasing blood flow to the tick bite-site (Dai et al., 2010). Immunization of mice with the recombinant protein interfered with tick feeding and decreased the spirochaete burden.

The Tick Salivary Lectin Pathway Inhibitor (TSLP, formerly P8) from salivary glands of I. scapularis was found to interfere with the lectin complement pathway and impair neutrophil phagocytosis and chemotaxis, and protects Borrelia from killing by those (Schuijt et al., 2011a).

Tick proteins were identified to be involved also in colonization of the vector and transmission of the intracellular bacterium Anaplasma phagocytophilum to vertebrate hosts. Generally, strategies of survival of these bacteria in the vector and transmission to vertebrate hosts are less explored than for Borrelia. A. phagocytophilum was found to induce expression of the I. scapularis salp16 gene in tick salivary glands during feeding. It was shown that RNA interference-mediated silencing of the salp16 gene expression diminished migration of the bacteria ingested via host blood meal to tick salivary glands, which demonstrates the specific requirement of the pathogen for a tick salivary protein to persist within the vector (Sukumaran et al., 2006). During the transmission of A. phagocytophilum to the vertebrate host, I. scapularis saliva probably modulates host inflammatory responses by inhibition of the production of inflammatory cytokines by macrophages during stimulation of Toll-like (TLR) and Nod-like receptor (NLR) signaling pathways (Chen et al., 2012).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.