The cattle tick Rhipicephalus (Boophilus) microplus is one of the most harmful parasites affecting bovines. Similarly to other hematophagous ectoparasites, R. microplus saliva contains a collection of bioactive compounds that inhibit host defenses against tick feeding activity. Thus, the study of tick salivary components offers opportunities for the development of immunological based tick control methods and medicinal applications. So far, only a few proteins have been identified in cattle tick saliva. The aim of this work was to identify proteins present in R. microplus female tick saliva at different feeding stages. Proteomic analysis of R. microplus saliva allowed identifying peptides corresponding to 187 and 68 tick and bovine proteins, respectively. Our data confirm that (i) R. microplus saliva is complex, and (ii) that there are remarkable differences in saliva composition between partially engorged and fully engorged female ticks. R. microplus saliva is rich mainly in (i) hemelipoproteins and other transporter proteins, (ii) secreted cross-tick species conserved proteins, (iii) lipocalins, (iv) peptidase inhibitors, (v) antimicrobial peptides, (vii) glycine-rich proteins, (viii) housekeeping proteins and (ix) host proteins. This investigation represents the first proteomic study about R. microplus saliva, and reports the most comprehensive Ixodidae tick saliva proteome published to date. Our results improve the understanding of tick salivary modulators of host defense to tick feeding, and provide novel information on the tick-host relationship.

Tick saliva serine protease inhibitors (serpins) facilitate tick blood meal feeding through inhibition of protease mediators of host defense pathways. We previously identified a highly conserved Amblyomma americanum serpin (AAS) 19 that is characterized by its reactive center loop being 100% conserved in ixodid ticks. In this study, biochemical characterization reveals that the ubiquitously transcribed AAS19 is an anti-coagulant protein, inhibiting the activity of five of the eight serine protease blood clotting factors. Pichia pastoris-expressed recombinant (r) AAS19 inhibits the enzyme activity of trypsin, plasmin and blood clotting factors (f) Xa and XIa, with stoichiometry of inhibition estimated at 5.1, 9.4, 23.8 and 28, respectively. Similar to typical inhibitory serpins, rAAS19 forms irreversible complexes with trypsin, fXa and fXIa. At a higher molar excess of rAAS19, fXIIa is inhibited by 82.5%, and thrombin (fIIa), fIXa, chymotrypsin and tryptase are inhibited moderately by 14 – 29%. In anti-hemostatic functional assays, rAAS19 inhibits thrombin but not ADP and cathepsin G activated platelet aggregation, delays clotting in recalcification and thrombin time assays by up to 250 s, and up to 40 s in the activated partial thromboplastin time assay. Given AAS19 high cross-tick species conservation, and specific reactivity of rAAS19 with antibodies to A. americanum tick saliva proteins, we conclude that rAAS19 is a potential candidate for development of a universal tick vaccine.

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We report the isolation of a keratinolytic-producing Bacillus subtilis strain and the characterization of the exceptional dehairing properties of its subtilisin-like keratinase. This enzyme can be an alternative to sodium sulfide, the major pollutant from tanneries, and may completely replace it. Its unique nonactivity upon collagen enhances its industrial potential.Enzymatic dehairing in tanneries has been envisaged as an alternative to sulfides (4,6,9,22,23). Tanneries are constantly concerned about the obnoxious odor and pollution caused by the extremely toxic sodium sulfide used in the dehairing process step (24). Deaths due to this toxic chemical process have even been reported (2,8). Worldwide, it is estimated that 315 million bovine leathers are produced per year. Considering a waste treatment cost of $0.30 per m 2 of leather produced (A. Klein, personal communication), more than $1 million is spent per day to treat the waste from tanneries around the world. We report here a novel keratinase from Bacillus subtilis that has the potential to replace sodium sulfide in the dehairing process.Microorganism isolation. Bovine hair, skins wastes, and soil samples were suspended and cultivated in a feather-broth medium (composition in grams per liter: delipidated feather meal [the sole carbon and nitrogen source], 10.0; NaCl, 0.5; K 2 HPO 4 , 0.3; and KH 2 PO 4 , 0.4 [pH 7.5]).The best keratinase-producing organism was identified as a B. subtilis strain (named strain S14) after classification based on homology (99%) of its 16S fragment with sequences from the NCBI databank by use of BLASTN 2.2.6 (1) (accession number AY345856).Keratinase production. The microorganism was cultivated in a 14-liter bioreactor in a culture medium composed of whey milk (a dairy byproduct containing 94.0% water, 5% lactose, 0.9% protein, and 0.1% fat), pH 8.5, and bovine hair (40 g/liter). A crude extract (supernatant) was obtained after centrifugation of the culture. Keratinolytic, subtilisin, and collagenase activities were assayed by using azokeratin, as described elsewhere (15). One unit of enzyme activity was defined as the amount of enzyme that increases absorbance by 0.1 per hour in the conditions described above (15).Dehairing assay. A fresh fleshed bovine hide was washed with a commercial detergent solution and cut into 15-by 5-cm pieces. Two hundred grams of skin (usually two pieces) was processed in a drum flask at 4 rpm with crude extract or water (control) in a proportion of 1.0 ml of liquid per g of skin. When necessary, pH was adjusted with lime. At the end of the process, the skin pieces were gently scraped with fingers to remove loose hairs. This procedure was necessary because rubbing in this laboratory-scale process was not as vigorous as in industrial drums. Total skin depilation was observed in the pH range from 7 to 10, with 4.8 U/g of skin. A complete depilation was reached in 9 h at pH 9.0, 24°C, with 4.8 U/g of skin.Samples of bovine skin were kept in contact with the crude extract, and the skin fragments were fi...

The tick Rhipicephalus (Boophilus) microplus is a blood-sucking ectoparasite of cattle that severely impairs livestock production. Studies on tick immunological control address mostly single-antigen vaccines. However, from the commercial standpoint, so far no single-antigen vaccine has afforded appropriate protection against all R. microplus populations. In this context, multi-antigen cocktails have emerged as a way to enhance vaccine efficacy. In this work, a multi-antigenic vaccine against R. microplus was analyzed under field conditions in naturally infested cattle. The vaccine was composed by three tick recombinant proteins from two tick species that in previous single-vaccination reports provided partial protection of confined cattle against R. microplus infestations: vitellin-degrading cysteine endopeptidase (VTDCE) and boophilus yolk pro-cathepsin (BYC) from R. microplus, and glutathione S-transferase from Haemaphysalis longicornis (GST-Hl). Increased antibody levels against three proteins were recorded after immunizations, with a distinct humoral immune response dynamics for each protein. Compared to the control group, a statistically significant lower number of semi-engorged female ticks were observed in vaccinated cattle after two inoculations. This reduction persisted for 3 months, ranging from 35.3 to 61.6%. Furthermore, cattle body weight gain was significantly higher in vaccinated animals when compared to control cattle. Compared to the single-antigen vaccines composed by VTDCE, BYC or GST-Hl, this three-antigen vaccine afforded higher protection levels against R. microplus infestations.