Dneasy blood and tissue kit

White sucker (n = 208) were collected from seven tributaries within the Great Lakes Basin between 2010 and 2012, including Areas of Concern (Figure 1). Great Lake drainages represented by these fish included that of Lake Michigan, Lake Superior and Lake Erie. Additionally, 11 white sucker were collected from two locations on the Athabasca River in Alberta Canada. Fish collection methods and IACUC are described elsewhere [13 (link),14 (link),15 (link)]. All fish were adults and greater than 350mm in length. Liver samples were preserved in RNAlater™. Liver or plasma were collected from these fish and stored at −80 °C until extraction of nucleic acids. Both liver and plasma were only collected from fish inhabiting the Sheboygan River. Plasma was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA), following the manufacturer instructions for nucleated blood. DNA from liver tissues preserved in ethanol was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA), following manufacturer instructions. DNA from RNAlater™ preserved liver tissue was also extracted using the DNeasy Blood and Tissue Kit, but following the Qiagen user developed protocol for purification of total DNA from soft tissues (http://www.qiagen.com/us/resources/download.aspx?id=7684840d-96bd-47a6-9d84-80cae16bf0e7&lang=EN&ver=3, accessed on 11 January 2021).

For DNA extraction from milk and NMC, 1 mL of samples were firstly centrifuged at 4 °C at 12,000× g for 30 min, and supernatant was removed together with fats by using a sterile cotton swab. Pellet was washed with 500 µL of 1X phosphate buffered saline (PBS) and resuspended in 200 µL of ATL buffer (Qiagen, Hilden, Germany) and proteinase K (Qiagen, Hilden, Germany) following the manufacturer’s instruction of the DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany). DNA was then extracted with the DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany) following the manufacturer’s instruction. For DNA extraction from the cheese, 10 g of sample were homogenized in a sterile stomacher bag with 90 mL of sterile Ringer solution (Oxoid, Milan, Italy) and mixed in a Stomacher 400 Circulator (Seward Ltd, Worthing, UK) at 300 rpm for 3 min. One mL of the first decimal dilution was transferred into 1.5 mL micro-tube and DNA was extracted by using the DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany).
Mycobiota was studied though the amplification of the D1 domain of the 26S rRNA gene using primers and condition described by Mota-Gutierrez et al. [17 (link)]. PCR amplicons were purified following the Illumina metagenomic pipeline (Illumina Inc. San Diego, CA, USA). Sequencing was performed with a MiSeq platform (Illumina), generating 250 bp paired-end reads.

For each dog, DNA was extracted from 100µL of EDTA blood using the Qiagen DNeasy Blood and Tissue Kit (Qiagen, Maryland, MD, USA) according to manufacturer’s instructions. The DNA was eluted in 200 µL elution buffer and stored at −20 °C until further analysis.
Before DNA extraction, individual ticks were washed in 70% ethanol, followed by 5% sodium hypochlorite, then rinsed with distilled water followed by a phosphate buffered saline (PBS) rinse for one minute. The ticks were then dried on sterile Whatman^® filter paper and placed into individually labelled sterile 2 mL Eppendorf tubes. Each tick was dissected into four parts with a sterile scalpel blade inside 2 mL Eppendorf tubes, using a new blade for each tick. A total of 180 µL of lysis buffer and 20 µL proteinase K (Qiagen, Maryland, MD, USA) was added to each tube then incubated at 56 °C overnight. Total DNA was then extracted using the Qiagen DNeasy Blood and Tissue Kit (Qiagen, Maryland, MD, USA), adjusted to 200 µL of buffer AE and stored at −20 °C until further use.
After extraction, DNA concentrations from the dog blood and ticks were determined by spectrophotometry (NanoDrop^® One C 2000 Spectrophotometer, Thermo Fisher Scientific, Madison, WI, USA). To minimise risk of contamination, DNA extractions, PCR preparation, PCR amplification and agarose gel electrophoresis were performed in separate rooms.