DNA Extraction
DNA from tissue, swab, and fecal samples were extracted using the Qiagen
DNEasyⓇ Blood and Tissue Kit (Qiagen, Inc). For the cloacal swab
samples, we used the manufacturer protocol for Purification of Total DNA
from Animal Blood or Cells with the optional pre-treatment for
Gram-positive bacteria lysis buffer incubation. For tissue and fecal
pellets, we included the optional lysis buffer incubation for 30 min at
37°C. After incubation, Buffer AL and proteinase K were added to the
tubes. The samples were then incubated at 56°C for 180 minutes (for
tissue) or 90 min (for feces) while shaking at 500 RPM (USA Scientific
Mixer HC- 8012-0000, Ocala, FL, USA). From here, the extraction was
completed according to the Purification of Total DNA from Animal Blood
or Cells protocol, beginning at Step 3 (addition of pure ethanol). An
extraction blank was included in each extraction. DNA in all samples and
blanks was quantified via Qubit to ensure extraction was successful.