Representatives of five ixodid tick genera were compared, both metastriate species (D. reticulatus, R. appendiculatus, H. excavatum and A. variegatum) and a prostriate species (I. ricinus). The D. reticulatus and I. ricinus ticks were collected by flagging the vegetation in selected locations of western Slovakia previously used for tick collecting; R. appendiculatus, H. excavatum and A. variegatum were obtained from colonies maintained at the Institute of Zoology (Bratislava). Hyalomma excavatum was the kind gift of Dr Michael Samish, Kimron Veterinary Institute,

Bait Dagan, Israel. It is both a two-host ditropic tick, with larvae and nymphs feeding on the same selleckchem individual host animal while adults feed on entirely different host species, and a three-host tick with larvae, nymphs and adults each feeding on a different animal. To maintain our H. excavatum colony, the ticks were fed on rabbits: 70–80% followed a two-host strategy while the remainder were three-host. Larvae fed for 6–9 days, nymphs for 7–10 days and adult females fed for 8–12 days to complete engorgement; larvae + nymphs (two-host strategy) completed engorgement as nymphs in 11–28 days. see more SGE was prepared by modifying the method of [13]. Briefly, at given times, ticks were gently removed from the laboratory animals and their

salivary glands dissected out in ice-cold sterile 0.15 m NaCl (0.9%) and washed three times in the same

solution. Salivary gland tissues were then homogenized and centrifuged at 10 000 g for 30 min at 4°C. Supernatant fluids were dried using a Speed-Vac, stored at 4°C and reconstituted in PBS before use. Pooled SGE was prepared from ticks feeding on laboratory rabbits for two time periods: 3 days representing the early (slow) period and 7 days representing Osimertinib molecular weight the late (rapid) phase of engorgement (Table 1). Before testing, the pooled dried SGE was diluted such that 10 μL contained SGE from a single tick. The hypostome of ticks is sclerotized and does not change size or shape once the tick has moulted [14]. Live ticks were immobilized on double-sided tape, and the tube-shaped hypostome from the apex to the base of the cheliceral shaft (dorsal aspect) was measured by means of an eyepiece and lens micrometre using a binocular microscope (Nikon SMZ 645; Optoteam S.R.O., Bratislava, Slovak Republic) at magnification, ×50. Antigrowth factor activities were measured using commercial ELISA kits and recombinant growth factors obtained from R&D Systems (Abingdon, UK): human fibroblast growth factor, FGF-2 (basic; DFB50); human hepatocyte growth factor, HGF (DHG00); human IL-6 (D6050); human keratinocyte growth factor, KGF (DKG00); human/mouse platelet-derived growth factor, PDGF-AA (DAA00); human stromal cell-derived factor, SDF-1α (DSA00); and human-transforming growth factor, TGF-β1 (DY240).