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.
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.
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