Tissuelyzer

Since the survey was part of a larger study in which both viruses and bacteria were examined, both RNA and DNA were isolated from the tick samples. After homogenization of the ticks using a QIAGEN TissueLyzer (Qiagen GmbH, Hilden, Germany), RNA extraction was performed in a QIAGEN M48 BioRobot using the MagAttract® RNA Tissue Mini 48kit. For cDNA synthesis, Illustra™ Ready-to-GO RT-PCR beads kit (GE Healthcare, UK) and random hexamer primers were used [20 ].
All tick cDNA samples were individually assayed using a real-time PCR targeting the citrate synthase (gltA) gene of Rickettsia spp. [24 (link)]. The reactions were run in a Rotor-Gene 3000 (Qiagen, Sydney, Australia) using LightCycler® TaqMan® Master (Roche Diagnostics, Mannheim, Germany). Two to five μl cDNA was used as a template in each reaction, together with 0.25 μl LC Uracil-DNA glycosylase (UNG) (Roche Diagnostics, Mannheim, Germany) to minimize the risk of contamination. In each amplification trial a negative control, sterile water and a positive standard plasmid constructed by cloning the PCR product into a PCR 4-TOPO vector (TOPO® TA Cloning® kit for Sequencing, Invitrogen, Carlsbad, CA, USA) and containing the cloned 74 bp fragment of the gltA gene were included in 10-fold serial dilutions.

For study purpose, one biopsy (15–20 mg) was taken from the descending duodenum of each patient. Fresh tissue samples were snap frozen and stored in liquid nitrogen until preparation. Frozen biopsies were disrupted and homogenized by TissueLyzer from Quiagen (Hilden, Germany). Total RNA was isolated using AllPrep^® DNA/RNA Micro kit (QIAGEN, Hilden, Germany) and stored at −70 °C.
Before microarray analysis RNA integrity and concentration was examined on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) using the RNA 6.000 LabChip Kit (Agilent Technologies) according to manufacturer’s instructions. Then, 250 ng RNA per sample was ethanol precipitated with GlycoBlue (Invitrogen) as carrier and dissolved at a concentration of 100–150 ng/µL prior to probe synthesis using the TargetAmp™- Nano Labeling Kit for Illumina Expression BeadChip (Epicentre Biotechnologies, Madison, WI, USA). From each probe, 750 ng of cRNA were hybridized to Human HT-12 v4 Expression BeadChips (Illumina, San Diego, CA, USA) and scanned on the Illumina HiScan instrument according to the manufacturer’s specifications.

For each condition we prepared three replicates of 20 pooled fly thorax-abdomens for NMR spectroscopy. Samples were directly lyophilized and stored at 4°C (Jensen et al., 2013 (link)). Materials were mechanically homogenized with a TissueLyzer (Quiagen) in 1 ml of ice-cold acetonitrile (50%) for 3×1 min. Hereafter samples were centrifuged (10,000 g) for 10 min at 4°C and the supernatant (900 μl) was transferred to new tubes, snap frozen and stored at −80°C. The supernatant was then lyophilized and stored at −80°C. Immediately before NMR measurements, samples were rehydrated in 200 ml of 50 mM phosphate buffer (pH 7.4) in D₂O, and 180 ml was transferred to 3 mm NMR tubes. The buffer contained 50 mg/l of the chemical shift reference 3-(trimethylsilyl)-propionic acid-D4, sodium salt (TSP), and 50 mg/l of sodium azide to prevent bacterial growth.

Approximately 5 mg of each sample of the PFC (n = 5/group) was homogenized in 100 μl of homogenization buffer (0.05% Tween 20 and protease inhibitor cocktail (Thermo Fisher Scientific Inc., Waltham, MA, USA) in PBS, pH 7.2). After 3 cycles of 1 min and 50 Hz in the tissuelyzer (Quiagen, Germantown, Maryland, USA), samples were centrifuged at 11000×g for 30 min at 4 °C. After centrifugation, supernatants were transferred to a new tube and stored at −80 °C until the assay. Total protein content of each sample was measured using the BCA protein assay kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). Levels of tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), interleukin 6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), interleukin 4 (IL-4), and interleukin 10 (IL-10) were measured by X-Map technology using a Milliplex MADPK-71K adipokine kit according to the manufacturer’s description (Merck Millipore, Spain).

Intestinal permeability was measured as described previously (8 (link)): After 6 days, mice were anaesthetized mice using isoflurane (Forene, Abbott, Wiesbaden, Germany). Following laparotomy, the colon was mobilized and opened at the ileocaecal valve and at the upper rectum. After flushing the colon with PBS at RT to remove blood and stool, the colon was ligated at the cutted ends without compromising the blood supply. To determine intestinal permeability 200µl of 4kDa FITC dextran diluted in PBS (1 mg/ml) was injected in the ligated colon. After 1h blood from the inferior vena cava was taken to measure the concentration of 4 kDa FITC dextran translocated from the colonic lumen into the blood. The blood samples were centrifuged at 13.400 rpm for 10 minutes at 4°C, and the luminescence of the serum was quantified by using Genios Pro Reader (Tecan, Maennedorf, Switzerland). After blood collection mice were euthanized by exsanguination, the colon was harvested and the length was measured.
The ligations of the colon were removed and the colon was cut longitudinally into two pieces. One part was fixed in 4% paraformaldehyde embedded in paraffin and sectioned. The other half of the colon was lyzed and homogenized with a Tissue Lyzer (Quiagen, Hilden, Germany) in a SDS lysis buffer and used for Western Blot analysis.

In order to determine if the timing of the infectious blood meal affected the within-mosquito viral kinetics, mosquitoes were sampled at 5, 8 and 11 days post-infection (dpi) to test for infection and dissemination across all groups (sample sizes are provided in Additional file 1: Table S1). Mosquito legs and bodies were placed into separate tubes containing 900 μl BA-1 diluent media and BBs. Samples were then homogenized twice at 25 Hz for 3 min using a Qiagen Tissuelyzer (Qiagen, Hilden, Germany). RNA was extracted (5× MagMax 96 Viral RNA Isolation Kit; Applied Biosystems, Foster City, CA, USA) and tested for the presence of viral RNA via qRT-PCR (SuperScript III One-Step RT-PCR System; Invitrogen, Carlsbad, CA, USA) as in [40 (link)]. Each treatment was repeated a total of three times and data was averaged over these replicates. Differences among the treatment groups for infection and dissemination rates were tested by a chi-square test for multiple proportions on 5, 8 and 11 dpi.