Dneasy 96 tissue kit

All adult, nymphal and if required, larval I. scapularis were sent to the National Microbiology Laboratory (NML) at Winnipeg (Public Health Agency of Canada, Winnipeg, Manitoba, Canada) for species verification. Adult and nymphal I. scapularis subsequently underwent testing at NML for B. burgdorferi, and four other I. scapularis-borne pathogens: Anaplasma phagocytophilum, B. miyamotoi, Babesia microti and Powassan Virus.
Laboratory analyses have been previously described [20 (link)]. Briefly, DNA was extracted using DNeasy 96 tissue kits (QIAGEN Inc., Mississauga, Canada). Initial screening for Borrelia spp. was conducted using the 23s ribosomal RNA real-time polymerase chain reaction (PCR) assay. This was coupled with the msp2 real-time PCR for the detection of A. phagocytophilum [21 (link)]. Samples that tested positive for Borrelia spp. were subjected to the ospA real-time PCR to detect B. burgdorferi and the IGS real-time PCR for B. miyamotoi. Borrelia miyamotoi-positive samples were then verified with the glpQ real-time PCR [22 (link)]. Real-time PCR for the CCTeta gene was used to detect B. microti [23 ]. To ensure contamination did not occur during extraction and PCR runs, water blanks were used.

Blacklegged ticks submitted to the NML through active and passive tick surveillance are routinely tested for DNA or RNA of A. phagocytophilum, Babesia microti, a variety of Borrelia species including B. burgdorferi, and Powassan encephalitis virus by real-time polymerase chain reaction (PCR) as previously described.^{37 (link),38 (link)} Briefly, for the detection of A. phagocytophilum DNA in ticks, we used Qiagen^® DNeasy 96 tissue kits (Qiagen Inc., Mississauga, ON) for DNA extraction as per the manufacturer’s instructions. We eluted DNA in 200 μL of AE buffer and stored at −80°C before use. We used a duplex real-time PCR assay to screen the samples for A. phagocytophilum by targeting the msp2 gene.^{39 (link)}We monitored each round of DNA extractions for cross-contamination by including at least two samples consisting only of nuclease-free water. Synthetic double-stranded DNA controls (Integrated DNA Technologies, Skokie, IL) for Anaplasma were included as positive controls in each PCR run, whereas no-template controls consisting of master mix only served as negative controls. In addition, our positive control DNA for A. phagocytophilum was an equine isolate (MN-93, courtesy of Tim Kurtti, University of Minnesota, MN) that had been propagated in HL-60 promyelocytic cell line (ATCC CCL-240).

Samples were conserved at room temperature for a maximum of 2 years. If a site had less than 30 ticks in a year, all samples were tested. If more than 30 ticks had been collected, a subsample of 30 to 40 ticks was randomly selected. Ticks from 2019 and 2020 were cut in half; one half was saved for the main study and the other was tested for the current investigation. Whole ticks were tested for the year 2021. In brief, DNA was extracted with DNeasy 96 tissue kits (Qiagen, Mississauga, ON, Canada) according to the manufacturer protocol. Samples were screened for Ap using multiplex real-time PCR (Courtney et al., 2004 (link)). Ap strains were identified with a single-nucleotide polymorphism assay targeting the 16S rRNA gene developed by Krakowetz et al. (2014 (link)).