Bullet blender storm

Frozen mosquito body samples were homogenized in a Bullet Blender Storm (Next Advance), according to a previously reported protocol (Fros et al., 2015b (link)). In total, 30 μl of the body tissue homogenate or mosquito saliva was added to one well of a 96-well plate containing Vero cells at 80% confluency. After incubation for 2 h at 37°C, the cell medium was replaced with fresh DMEM HEPES complete medium, and the cells were kept at 37°C for another 6 days. Positive viral infection was determined by checking for CPE at both 3 dpi and 6 dpi for each well. The infection rate and transmission efficiency were expressed as the percentage of virus-positive mosquito bodies or saliva over the total number of mosquitoes used for the salivation assay. Viral titers were determined by EPDAs using six and four bodies and saliva samples of the USUV-NL and USUV-IT infected mosquitoes, respectively.

Organs and tissues were snap frozen on dry ice and stored at −80°C. For DNA extraction, samples were thawed and immediately homogenized in at least 200 μl lysis buffer (4 M urea, 200 mM Tris, 20 mM NaCl, 200 mM EDTA, pH 7.4) per 50 mg tissue using a BulletBlender Storm instrument (Next Advance). The tissue suspension was then incubated overnight at 37°C with 0.6 mg proteinase K. DNA was extracted using the High Pure PCR product purification kit (Roche). Real-time PCR reactions were prepared using the QuantiTect SYBR Green PCR Kit (Qiagen) and run on a RotorGene 3000 instrument. Each reaction contained 50 ng DNA and 0.5 μM of each primer. T. cruzi-specific primers TCZ-F and TCZ-R (Cummings and Tarleton, 2003 (link)), targeted at the 195 bp satellite repeat, and mouse-specific primers GAPDHf and GAPDHr (Cencig et al., 2011 (link)), targeted at the murine gapdh gene, were used. Measurements of T. cruzi DNA content were normalized using the ratio of Ct values for T. cruzi- and mouse-specific PCRs, and converted to estimated numbers of parasite equivalents by reference to a standard curve with a range of 2.5 × 10⁶–2.5 × 10⁻¹ parasite equivalents ml⁻¹. The standard curve was established from serial dilution of a DNA sample derived from 75 mg homogenized muscle tissue, spiked with 2 × 10⁷ epimastigotes, using DNA from unspiked equivalent samples as the diluent.

The SDC method of protein extraction was adapted from a previously published protocol (Pasing et al., 2017 (link)). Blubber was processed in two batches of ∼50 mg, which were minced with a sterile scalpel on ice and added to 500 µl SDC Lysis Buffer [1% w/v SDC, 8 M urea, 5 mM dithiothreitol (DTT) in 50 mM ammonium bicarbonate] in a Navy RINO^® bead tube (Next Advance Inc., USA). Samples were homogenized in the Bullet Blender Storm^® instrument (Next Advance Inc., USA) for two cycles of 2 min each at power 10, with 1 min of cooling on ice between cycles. Homogenates were further disrupted by sonication for three cycles, 15 s each, at 4 watts using a hand-held sonicator (VirSonic 60, Virtis, USA) and centrifuged to pellet insoluble cell debris and separate lipids. Tissue homogenates were extracted from under the top lipid layer and transferred to clean tubes. To remove any remaining lipids, four volumes of methanol and one volume of chloroform were added to homogenate aliquots, mixed, and centrifuged. The top layer containing lipids was removed, and four volumes of methanol were added to precipitate proteins. Protein pellets were air dried after centrifugation.

After dissection, parietal bones were homogenized in RNAse free tubes with beads (Navye1-RNA) with TRIzol using the Bullet Blender Storm homogenizer (Next Advance). After chloroform step total RNA isolation was performed according to protocol provided by manufacturer (RNAeasy kit). After DNAse treatment, RNA was synthesized into cDNA as previously described.

All animal experiments were performed in compliance with guidelines set out by the Canadian Council on Animal Care. All animal protocols (protocol 2017-028) were reviewed and approved by the University of Western Ontario Animal Use Subcommittee, a subcommittee of the University Council on Animal Care. Six- to 8-week-old female BALB/c mice (Charles River Laboratories) were injected via tail vein with 100 μl of bacterial culture containing 1 × 10⁷ CFU of bacteria. To prepare the bacteria, strains were grown to an OD₆₀₀ 2 to 2.5 in TSB, washed twice with PBS, and resuspended to the desired numbers in PBS. Infections were allowed to proceed for up to 96 h before animals were euthanized or when they met guidelines for early euthanasia. Organs were harvested in PBS plus 0.1% (vol/vol) Triton X-100 and homogenized in a Bullet Blender Storm (Next Advance, Troy, NY), using 2 runs of 5 min at setting 10 and metal beads. Dilutions of organ homogenates were plated on tryptic soy agar (TSA) for CFU enumeration.

Participants were provided with a Fe-Col^® (Alpha laboratories, Hampshire, United Kingdom) fecal sample collection kit to collect stool samples. Stool samples were collected once and were transported on ice to the laboratory where they were stored at −80°C until analysis. DNA was extracted using the commercial EZNA Stool DNA Kit (Omega Biotek, Madrid, Spain), following the manufacturer’s instructions, with a bead-beating homogenizer (Bullet BlenderStorm, Next Advance, New York, NY, United States) using glass beads for 3 min at speed 10 (following the manufacturer’s recommendations). The elution volume was 100 μL. The DNA concentration and purity of was measured using the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific, Waltham, MA, United States). Microbiota analyses were performed by amplifying the V3-V4 hypervariable regions of the bacterial 16S rRNA gene. The 459 base pair (bp) amplicon was visualized in a 0.8% agarose gel stained with ethidium bromide. The bands were cut and cleaned using the MinElute Gel extraction kit (Qiagen, Hilden, Germany). DNA amplicons were sequenced on a MiSeq Illumina platform (Illumina, San Diego, CA, United States). Raw data in fastq format have been deposited and are publicly available with the accession number PRJNA564612 the NCBI Biosample database¹.