Evasin-3-like anti-chemokine activity in salivary gland extracts of ixodid ticks during blood-feeding: a new target for tick control.

Ticks exploit many evasion mechanisms to circumvent the immune control of their hosts including subversion of the communication language between cells of the immune system provided by chemokines and other cytokines. One subversive molecule secreted in the saliva of Rhipicephalus sanguineus is Evasin-3, a structurally unique 7 kDa protein that selectively binds the neutrophil chemoattractants, CXCL8 and (with lower affinity) CXCL1. We compared anti-human CXCL8 and anti-mouse CXCL1/KC activities in salivary gland extracts prepared from adult Amblyomma variegatum, Rhipicephalus appendiculatus and Dermacentor reticulatus ticks during blood-feeding. Both anti-CXCL8 activity and anti-CXCL1 activity were detected in all species and in both adult females and males, with consistently higher activity levels against CXCL8. These results suggest that Evasin-3-like activity is common amongst metastriate ixodid tick species, and provide further evidence of the importance to ticks in controlling neutrophils during blood-feeding. As such, Evasin-3 offers a new target for anti-tick vaccine development.

Manipulation of host cytokine network by ticks: a potential gateway for pathogen transmission.

Ticks are obligatory blood-feeding arthropods that secrete various immunomodulatory molecules to antagonize host inflammatory and immune responses. Cytokines play an important role in regulating these responses. We investigated the extent to which ticks interact with the sophisticated cytokine network by comparing the effect of salivary gland extracts (SGE) of 3 ixodid tick species, Dermacentor reticulatus, Amblyomma variegatum and Ixodes ricinus, all of which are important vectors of tick-borne pathogens. Using specific ELISAs, anti-cytokine activity was demonstrated with 7 cytokines: IL-8, MCP-1, MIP-1alpha, RANTES, eotaxin, IL-2 and IL-4. The results varied between species, and between adult males and females of the same species. Relatively high activity levels were detected in saliva of female D. reticulatus, confirming that the observed anti-cytokine activities are an integral part of tick saliva secreted into the host. Results with fractionated SGE indicated that from 2 to 6 putative cytokine binding molecules are produced, depending on species and sex. Binding ability of SGE molecules was verified by cross-linking with radio-isotope labelled MIP-1alpha. By targeting different cytokines, ixodid ticks can manipulate the cytokine network, which will greatly facilitate blood-feeding and provide a gateway for tick-borne pathogens that helps explain why ticks are such efficient and effective disease vectors.

Vesicular stomatitis virus nucleocapsid protein production in cells treated with selected fast protein liquid chromatography fractions of tick salivary gland extracts.

A salivary gland extract (SGE) prepared from 5-days-fed Dermacentor reticulatus female ticks was fractionated by fast protein liquid chromatography (FPLC). The effect of three FPLC fractions selected on the basis of anti-interleukin 8 (anti-IL-8) activity on vesicular stomatitis virus (VSV) nucleocapsid (N) protein formation in mouse L-cells was determined. Infected 14C-labeled cells treated with the FPLC fractions were analyzed by two-dimensional (2D) electrophoresis. The yields of VSV N protein were evaluated by Imagemaster software analysis. Most noticeable was an increase in the N protein production after treatment with the fraction 39 corresponding to the major peak of the anti-IL-8 activity. The nature of the substance in SGE that was responsible for this effect remains unclear.

Heterogeneity in the effect of different ixodid tick species on human natural killer cell activity.

Tick saliva plays a vital role in blood-feeding, including manipulation of the host response to tick infestation. Furthermore, a diverse number of tick-borne pathogens are transmitted to vertebrate hosts via tick saliva, some of which exploit the immunomodulatory activities of their vector's saliva. We report that salivary gland extracts (SGE) derived from Dermacentor reticulatus adult ticks induce a decrease in the natural killer (NK) activity of effector cells obtained from healthy human blood donors. The decrease was observed with SGE from both female and male D. reticulatus fed for either 3 or 5 days on mice, but no significant effect was observed with SGE from unfed ticks or ticks that had fed for 1 day. These results indicate that the tick anti-NK factor(s) is only active after blood-feeding has commenced. Microscopic examination revealed that the first step of NK activity, namely effector/target cell conjugate formation, was affected by SGE. The observed reduction in conjugate formation occurred when effector (but not target) cells were treated with SGE for 30 min, and the effect persisted after 12 h of treatment. Similar but less potent anti-NK activity was detected for SGE from Amblyomma variegatum and Haemaphysalis inermis. By contrast, SGE derived from Ixodes ricinus and Rhipicephalus appendiculatus female ticks did not decrease NK activity. The apparent absence of such activity in these two important vectors of tick-borne viruses suggests that control of NK cells does not play an important role in promoting virus transmission, at least for these particular species.

Anti-interleukin-8 activity of tick salivary gland extracts.

Interleukin-8 (IL-8) is one of many mammalian chemokines (chemotactic cytokines) that direct mammalian inflammatory and immune cells to sites of injury and infection. Chemokines are produced locally and act on leucocytes through selective receptors. The principal role of IL-8 is to control the movement and activity of neutrophils. To date, several tick species have been shown to modulate the production or activity of certain cytokines but none of these are chemokines. Using an IL-8 specific ELISA, we showed that salivary gland extracts (SGE) from several ixodid tick species (Dermacentor reticulatus, Amblyomma variegatum, Rhipicephalus appendiculatus, Haemaphysalis inermis and Ixodes ricinus) reduced the level of detectable IL-8. Analyses of fractionated SGE revealed one similar peak of activity for D. reticulatus, A. variegatum and R. appendiculatus; a second peak, observed for D. reticulatus and A. variegatum, differed between the two species. Using radiolabelled IL-8, SGE and peak activity fractions of D. reticulatus were shown to bind the chemokine, and to inhibit binding of IL-8 to its receptors on human granuolocytes enriched for neutrophils. The biological significance of these observations was demonstrated by the ability of SGE to inhibit IL-8 induced chemotaxis of human blood granulocytes. Future isolation and characterization of the active molecules will enable determination of their functional roles in bloodfeeding and effect on tick-borne pathogen transmission.

Inhibition of the antiviral action of interferon by tick salivary gland extract.

The saliva of haematophagous arthropods (e.g. mosquitoes, sandflies and ticks) contains potent immunomodulatory activities that counter their hosts' haemostatic, inflammatory and immune responses to facilitate blood-feeding. Such effects are exploited by arthropod-transmitted pathogens to promote their transmission. We investigated the ability of tick saliva to enhance arthropod-borne virus (arbovirus) transmission by determining its effect on the antiviral action of murine interferon (IFN alpha/beta). Salivary gland extract (SGE) was prepared from partially fed adult female Dermacentor reticulatus ticks that had been feeding on mice for either 3 or 5 days (SGED3 and SGED5, respectively). We demonstrated that SGE inhibits the antiviral effect of IFN as measured by a biological assay using vesicular stomatitis virus (VSV), and by two-dimensional electrophoretic analysis of the appearance of selected VSV proteins. The most pronounced effect was observed when mouse L cells were treated with SGE prior to IFN treatment. Following pretreatment with SGE, virus multiplication (which was fully blocked by IFN treatment alone) achieved yields similar to those obtained from infected cells not treated with IFN. Contemporaneous treatment, or treatment with SGE after IFN, was less effective. In parallel with these findings, formation of early viral proteins, N (nucleocapsid protein) and P (phosphoprotein), which was blocked by IFN, was detectable following pretreatment with SGE. The ability to inhibit the antiviral action of IFN was higher for SGED3 compared to SGED5. Demonstration that tick SGE can promote virus replication by suppressing the action of IFN helps explain why ticks are such efficient vectors of arboviruses.

Promotion of vesicular stomatitis virus nucleocapsid protein production by arthopod saliva.

In a previous study (Hajnicka, V. et al., Parasitology 116, 533-538, 1998), the infectivity titer of vesicular stomatitis virus (VSV) was shown to increase up to 10,000-fold when mouse L cells were treated with tick salivary gland extract (SGE) prior to infection. To examine this effect at the level of viral protein production, radiolabeled VSV-infected cells were analyzed by double-dimensional gel electrophoresis. A pre-treatment of cells with SGE from partially fed ticks in amounts corresponding to 1 or 3 salivary glands increased the level of both viral nucleocapsid (N) protein and phosphoprotein (P) in a dose-dependent manner. The effect was more pronounced for N protein and could account for the dramatic increase in infectious virus yield. Promotion of viral infectivity by arthropod saliva may support the arthropode-borne transmission cycle of VSV.

Tick salivary gland extracts promote virus growth in vitro.

Saliva of blood-feeding arthropods promotes infection by the vector-borne pathogens they transmit. To investigate this phenomenon in vitro, cultures of mouse L cells were treated with a salivary gland extract (SGE) prepared from feeding ticks and then infected with vesicular stomatitis virus (VSV). At low input doses of VSV, viral yield was increased 100-fold to 10,000-fold by 16-23 h post-infection compared with untreated cultures, and depending on the SGE concentration. SGE-mediated acceleration of viral yield corresponded with the earlier appearance of VSV nucleocapsid protein as detected by 2-dimensional electrophoresis of infected cells. The observation that physiological doses of virus (i.e. doses likely to be inoculated by an infected arthropod vector into its vertebrate host during blood-feeding) respond to SGE treatment in vitro provides a new opportunity for identifying the factors in tick saliva that promote virus transmission in vivo.