Ticks and DNA/RNA/protein extraction
Questing and feeding Rhipicephalus spp. adult female ticks were collected in 27 farms located in different Sicilian regions (Palermo, Enna, Messina, Siracusa and Trapani). A total of 300 ticks were collected and analyzed for this study. Of them, 12 were questing ticks and 288 were fully engorged ticks collected from sheep, goats or dogs. These ticks were collected from adult animals living in pathogen endemic areas, thus likely to have chronic infections. Ticks were identified using morphological keys for the Italian Ixodidae . The ticks were incubated for three days in the laboratory prior to dissection and RNA/DNA/protein extraction. Individual ticks were dissected and whole internal organs extracted and used for DNA, RNA and protein extraction using TriReagent (Sigma, St. Louis, MO, USA) following manufacturers recommendations. Animal experiments were conducted with the approval and supervision of the Intituto Zooprofilattico Sperimentale della Sicilia Institutional Animal Care and Use Committee (project IZS SI 10-06).
Identification of pathogen infection in naturally infected ticks
The DNA was resuspended in sterile distilled water and stored at -20°C until used. For the initial screening, PCR analyses for Anaplasma, Ehrlichia and Rickettsia spp. were performed as described previously  with 1 μl (0.1-10 ng) DNA using 10 pmol of each primer and the Ready-To-Go PCR beads (Amersham, Piscataway, NJ, USA). Reactions were performed in an automated DNA thermal cycler for 35 cycles. PCR products were electrophoresed on 1% agarose gels to check the size of amplified fragments by comparison to a DNA molecular weight marker (1 Kb DNA Ladder, Promega, Madison, WI, USA). Control reactions were done without the addition of DNA to the reaction to rule out contaminations during PCR. Reverse line blot (RLB) was used for detection of Babesia and Theileria spp. as described previously .
To confirm pathogens in ticks, PCR and sequence analysis of cloned amplicons were performed for Anaplasma, Ehrlichia and Rickettsia spp. Amplified fragments were resin purified (Promega), cloned into pGEM-T vector (Promega) and sequenced in an accredited service laboratory (BaseClear, Leiden, The Netherlands) using vector specific primers. The BLAST tool was used to search the NCBI databases in order to identify sequences reported previously with identity to sequences obtained herein. Gene sequences were deposited in the GenBank with accession numbers GQ857075-GQ857078
. RLB was used to confirm T. annulata infection.
Two-Dimensional Difference in Gel Electrophoresis (2-D DIGE)
Following protein extraction with TriReagent (Sigma), protein samples were purified using a 2-D Clean-Up Kit (GE Healthcare, Madrid, Spain) according to the manufacturer's instructions to remove any contaminant substances that could interfere with the 2-D DIGE procedure. The protein pellet was resuspended in 25 μl of 2-D lysis buffer (7 M urea, 2 M thiourea, 4% w/v CHAPS, 25 mM Tris-HCl, pH 8.0) and protein concentration was determined using the 2D-Quant Kit (GE Healthcare).
In order to reduce variance from individual-to-individual variation and to obtain enough protein quantity to perform the experiment, protein samples from the same experimental group were pooled. Consequently, a total of 8 different samples were analyzed by 2D-DIGE, four infected with R. conorii, E. canis, T. annulata or A. ovis and their respective uninfected controls of the same tick species and collected from the same type of host or off-host.
CyDye DIGE fluor labeling kit for scarce protein samples (GE Healthcare) was used to label tick proteins according to the manufacturer's protocol. Briefly, for cysteine reduction before labeling, 5 μg of protein of each sample were incubated with 2 nmol Tris (2carboxyethyl) phosphine hydrochloride (TCEP; Sigma) at 37°C for 1 hour in the dark and, for labeling, 4 nmol of Cy5 dye in 2 μl of anhydrous DMF (Sigma) were added and the samples were incubated at 37°C for 30 min in the dark. For internal standardization, a pool of equal amounts of all samples (5 μg per sample) was created and labeled with Cy3 dye with the same procedure but scaling adjusting the quantities of reagents according to the amount of protein (40 μg). The reaction was quenched by adding and equal volume of 2 × sample buffer (7 M urea, 2 M thiourea, 4% w/v CHAPS, 1% v/v IPG buffer pH 3-11, 0.2% w/v DTT). Before 2-D separation, 5 μg of the Cy3-pool was mixed with 5 μg of each sample.
For the first dimension, 24-cm 3-11 NL pH range IPG strips were rehydrated overnight in 450 μL of DeStreak Rehydration Solution (GE Healthcare) supplemented with 0.5% IPG buffer pH 3-11 (GE Healthcare) using a reswelling tray. IEF was performed at 20°C using an Ettan IPGphor 3 (GE Healthcare). Samples were applied using anodic cup loading and the isoeletrofocusing was carried out using the following conditions: 300 V for 3 h, 300-1000 V for 6 h, 1000-10000 V for 3 h, 10000 V for 3 h and 500 V for 3 h. Prior to second dimension, focused IPG strips were incubated for 10 min equilibration buffer containing 50 mM Tris-HCl pH 8.8, 6 M urea, 30% v/v glycerol, 2% w/v SDS, 0.5% w/v DTT and traces of bromophenol blue. Equilibrated IPG strips were placed onto 12% homogeneous SDS-polyacrylamide gels casted in low fluorescence glass plates using an Ettan-DALT Six System (GE Healthcare). Electrophoresis was carried out at 20°C and 0.5 W/gel for 30 min followed by a second step at 15 W/gel for 4 hours.
Image acquisition and data analysis
Proteins were visualized using an Ettan DIGE Imager (GE Healthcare) following the manufacturer's instructions. Image analysis was performed with DeCyder 2 D Software, version 7.0 (GE Healthcare). Sixteen images were considered for the analysis, 8 corresponded to the different samples labeled with Cy5 and 8 corresponded to sample pool labeled with Cy3 and acquired individually with each gel. Spot co-detection, normalization of each spot against the corresponding value of the internal pool and volume ratios calculation were carried out using Differential In-Gel Analysis (DIA) module. In the Biological Variation Analysis (BVA) module, the 16 spot maps were distributed in 9 groups, that is, standard, and the 8 different samples (4 controls and 4 infected) and the standard image most representative with average quality was assigned as the master image. After matching of images, average ratios between groups were calculated. Protein spots with 5-fold as threshold in the average ratio were considered as differentially expressed between the samples under comparison.
Selection and preparation of protein samples for mass spectrometry
For preparative gels, equal amounts of all samples were pooled. Due to sample limitation, only 8 μg of each sample were mixed obtaining 64 μg of total proteins. 2-D electrophoresis was carried out in the same conditions as described above for CyDye labeled samples, but in this case, after second dimension, the gel was stained with Sypro Ruby (Molecular Probes, Invitrogen, Eugene, OR, USA) following the protocol recommended by the manufacturer. Proteins were visualized by fluorescence using an Ettan DIGE Imager (GE Healthcare) selecting 100 μm as pixel size and channel Sypro Ruby 1 with 0.4 of exposure to acquire the gel image. The gel was matched automatically in the BVA module of DeCyder software with the DIGE images. Of all spots matching with this gel, only those spots that were identified as differentially expressed and appeared at least in two of the four tick-pathogen groups under comparison were selected for mass spectrometry analysis. The 2-D electrophoresis stained gel was washed twice for 10 min with distilled water. Selected protein spots were visualized with an UV benchtop transilluminator (UVP, Cambridge, UK), manually excised from the gels, dehydrated with acetonitrile and vacuum-dried (Savant Speed Vac, mod SPD, 121 P, equipped with a vacuum pump OFP-400). After drying, spots were re-hydrated and digested in situ with trypsin (Promega) as described by Shevchenko et al.  with minor modifications. Stained protein gel spots were incubated in 50 mM NH4HCO3 with trypsin (5 ng/μl) for 1 hr in an ice-bath. The digestion buffer was removed and gels were covered again with 50 mM NH4HCO3 and incubated at 37°C for 12 hr. Whole supernatants were allowed to dry and then stored at 20°C until mass spectrometry analysis.
Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis
Peptide mass fingerprinting was conducted as previously described  using an Autoflex™(Bruker Daltonics, Bremen, Germany) mass spectrometer in a positive ion reflector mode employing 2, 5-dihydroxybenzoic acid as matrix and an AnchorChip™surface target (Bruker Daltonics). Peak identification and monoisotopic peptide mass assignation were performed automatically using Flexanalysis™software, version 2.2 (Bruker Daltonics). Database searches were performed using MASCOT http://matrixscience.com
 against the NCBI non-redundant protein sequence database http://www.ncbi.nih.gov. The selected search parameters were as follows: tolerance of two missed cleavages, carbamidomethylation (Cys) and oxidation (Met) as fixed and variable modifications, respectively, and setting peptide tolerance to 100 ppm after close-external calibration. A significant MASCOT probability score (p < 0.05) was considered as condition for successful protein identification.
Reverse phase-liquid chromatography (RP-LC)-MS/MS analysis
When peptide mass fingerprinting failed to identify a spot, the protein digest was dried, resuspended in 7 ul of 0.1% formic acid and analyzed by RP-LC-MS/MS in a Surveyor HPLC system coupled to an ion trap Deca XP mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The peptides were separated by reverse phase chromatography using a 0.18 mm × 150 mm BioBasic C18 RP column (Thermo Fisher Scientific), operating at 1.8 μl/min. Peptides were eluted using a 50-min gradient from 5 to 40% solvent B (Solvent A: 0,1% formic acid in water, solvent B 0,1% formic acid, 80% acetonitrile in water). ESI ionization was done using a microspray "metal needle kit" (Thermo Fisher Scientific) interface. Peptides were detected in survey scans from 400 to 1600 amu (8 μscans), followed by three data-dependent MS/MS scans, using an isolation width of 3 amu, normalized collision energy of 30%, and dynamic exclusion, applied during 3-min periods.
Peptide identification from raw data was carried out using the SEQUEST algorithm (Bioworks Browser 3.2, Thermo Fisher Scientific) and the PHENYX 2.6 search engine (GENEBIO, Switzerland). Database search was performed against the Apicomplexa, α-proteobacteria and metazoa databases downloaded from the Protein Knowledgebase (UniProtKB) http://www.uniprot.org. The following constraints were used for the searches: tryptic cleavage after Arg and Lys, up to two missed cleavage sites, and tolerances of 2 Da for precursor ions and 0.8 Da for MS/MS fragment ions and the searches were performed allowing optional Met oxidation and fixed Cys carbamidomethylation.
If the SEQUEST and PHENYX searches did not yield any positive results, high-quality spectra that had not been assigned to any protein identification were selected and manual de novo interpretation was conducted. These results were confirmed with PEAKS Studio 4.5 software (Bioinformatics Solutions Inc., Waterloo, ON, Canada).
Analysis of mRNA levels by real-time RT-PCR in naturally infected ticks
The genes encoding for differentially expressed unknown larval (Genbank accession number EF675686) and guanine nucleotide-binding (DQ066296) proteins were selected for mRNA analysis by real-time RT-PCR. mRNA levels were characterized in individual whole ticks naturally-infected with different pathogens using sequence-specific oligonucleotide primers (unknown larval protein, UNLP-F: 5'-TCATCCTCTGTGTGCTCGTC and UNLP-R: 5'-TCTCGAGGCAAGTGTCAATG; guanine nucleotide-binding protein, GNBP-F: 5'-GGGACTTGGAGGGCAAGAG and GNBP-R: 5'-ACACCTGCCAGACCCTGAT) as described previously . In all cases, matching groups of uninfected tick samples were analyzed concurrently for comparison. Real-time RT-PCR was done using the QuantiTec SYBR Green RT-PCR kit (Qiagen, Valencia, CA, USA) and a Bio-Rad iQ5 thermal cycler (Hercules, CA, USA) following manufacturer's recommendations. mRNA levels were normalized against tick 16 S rRNA using the comparative Ct method .