Gelstar nucleic acid stain

Plasmid DNA was isolated from a subset of strains that had varying AST and/or PFGE profiles using the Qiagen Miniprep kit (Qiagen Inc., Valencia, CA, USA) following the manufacturer’s instructions. The plasmid DNA was separated on 0.7% LE agarose gels (Lonza, Rockland, ME, USA) in 1X TBE buffer (Bio-Rad) and stained with GelStar nucleic acid stain (Lonza). The plasmid sizes were determined by comparing with BAC-Tracker ladder (8 to 165 kb range: Epicentre, Madison, WI, USA) and exACTGene 1 kb DNA ladder (Fisher Scientific, Pittsburgh, PA, USA) for smaller plasmids.
Plasmids were also characterized using a PCR-based replicon typing method, which was used to predict the plasmid incompatibility (Inc) groups using a previously published protocol [47 (link)], with positive controls provided by Alessandra Carattolli [48 (link)]. An aliquot (10 μL) of the amplified PCR product was loaded into a well of a 2% E-gel 48 with ethidium bromide (Invitrogen) and electrophoresed for 20 to 30 min along with the exACTGene 100-bp DNA ladder (Fisher Scientific) for size determination.

Unfed ticks were subjected to an ammonium hydroxide DNA extraction protocol as described previously [30 (link)], whereas DNA from fed ticks was extracted using the Qiagen DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA) following the manufacturer’s protocol for animal tissue. Ticks were initially pooled according to bird host; however, upon discovering the relatively high prevalence, we decided to process ticks individually. DNA was stored at −20 °C until PCR was performed. Conventional PCR was used to detect the 18S ribosomal RNA (rRNA) gene of the genus Babesia, with 25 µL reaction mixes containing 2.5 µL each of 5 µM primers BJ1 (5′-GTC-TTG-TAA-TTG-GAA-TGA-TGG-3′) and BN2 (5′-TAG-TTT-ATG-GTT-AGG-ACT-ACG-3′) [31 ], 5 µL nuclease-free water, 12.5 µL Green Go Taq (Promega, Madison, WI, USA), and 2.5 µL of DNA at 8–10 ng/µL. PCR was performed under thermal cycling conditions of initial denaturation at 94 °C for 10 min, 35 cycles of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 2 min with a final extension at 72 °C for 5 min, and then held at 4 °C. Amplified DNA was visualized with UV transillumination of a 1% agarose gel containing GelStar nucleic acid stain (Lonza, Rockland, ME, USA). Amplicons of 400–500 base pairs (bp) were excised from the gel and prepared for DNA sequencing using a gel extraction kit (QIAamp DNA Kit, Qiagen, Valencia, CA, USA).

To extract DNA from unfed ticks, an ammonium hydroxide protocol (unfed ticks) [35 (link)], or the Qiagen DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA) following the manufacturer’s protocol for animal tissue was used. The resulting DNA was stored at −20 °C until PCR was performed. Amplification of the 18S rRNA gene of Babesia was performed as previously described using the BJ1 (5′-GTC-TTG-TAA-TTG-GAA-TGA-TGG-3′) and BN2 (5′-TAG-TTT-ATG-GTT-AGG-ACT-ACG-3′) primers [43 ]. Amplicons were visualized by UV transillumination on a 1% agarose gel containing GelStar nucleic acid stain (Lonza, Rockland, ME, USA), and those that were 400−500 nucleotides in length, were excised from the gel, and prepared for DNA sequencing to confirm Babesia presence and species using the QIA amp DNA Kit (Qiagen, Valencia, CA, USA). DNA sequencing was performed at the University of California Davis Sequencing facility using the Big Dye Terminator cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) and PCR primers.