Molecular Screening of Tick-Borne Pathogens

A subset of adult and nymphal I. scapularis were submitted from each site to the National Microbiology Laboratory at Winnipeg (Public Health Agency of Canada, Winnipeg, Ontario). One hundred and sixty-seven adults of the spring cohort, 58 adults of the fall cohort and 60 nymphs were submitted from TP, while 19 adults of the spring cohort, 99 adults of the fall cohort and 11 nymphs were submitted from MP.
All samples were tested for B. burgdorferi, B. miyamotoi, Anaplasma phagocytophilum and Babesia microti. Laboratory analyses have been previously described [17 (link)]. In brief, DNeasy 96 tissue kits were used to extract DNA (QIAGEN Inc. Mississauga, Canada). The 23s ribosomal RNA real-time polymerase chain reaction (PCR) was then used to screen for Borrelia spp. If a sample tested positive, it was analyzed with the ospA real-time PCR to detect B. burgdorferi and the IGS real-time PCR to detect B. miyamotoi. The glpQ real-time PCR was then used to verify all B. miyamotoi-positive samples [18 (link)]. For A. phagocytophilum, the msp2 real-time PCR was employed [19 (link)], while the real-time PCR for the CCTeta gene was used to detect B. microti [20 ]. To verify that contamination did not occur during PCR runs, water blanks were used as negative controls.