A total of 4938 I. scapularis ticks collected in passive surveillance in 2012 (excluding the 68 used in validation) were tested using the developed testing protocol (Figure
1 ). The screening 23S and confirmatory ospA real-time PCR were used to assess B. burgdorferi infection as described above. The screening 23S PCR is a multiplex assay that also detects the presence of A. phagocytophilum DNA using primers specific for the msp2 gene (ApMSP2f and ApMSP2r)
[14 (link)]. Confirmation of infection with A. phagocytophilum is achieved by an in-house real-time PCR assay targeting 16S rRNA. The Borrelia miyamotoi-specific IGS real-time PCR was used to detect B. miyamotoi infections that were subsequently confirmed by B. miyamotoi glpQ real-time PCR. All real-time PCR assays were conducted using the conditions described above for B. miyamotoi IGS. Tick extracts that were positive for Borrelia spp. infection in the screening 23S real-time PCR, but negative for B. burgdorferi in the ospA real-time PCR, and negative in the B. miyamotoi IGS assay, were tested by 16S-23S IGS nPCR with the aim of sequencing products to identify other infecting Borrelia species.
Associations of infections and co-infections in ticks with province of origin, level of engorgement of the tick, host of origin, and tick instar were investigated by logistic regression in STATA version 11.0 for Windows (STATACorp, College Station, TX, USA). The most parsimonious multivariable model was created by backwards and forwards elimination and substitution of variables. Logistic regression models were used to investigate whether or not there were significant associations between infections of ticks with different pathogens. The level of significance throughout was P < 0.05.
[14 (link)]. Confirmation of infection with A. phagocytophilum is achieved by an in-house real-time PCR assay targeting 16S rRNA. The Borrelia miyamotoi-specific IGS real-time PCR was used to detect B. miyamotoi infections that were subsequently confirmed by B. miyamotoi glpQ real-time PCR. All real-time PCR assays were conducted using the conditions described above for B. miyamotoi IGS. Tick extracts that were positive for Borrelia spp. infection in the screening 23S real-time PCR, but negative for B. burgdorferi in the ospA real-time PCR, and negative in the B. miyamotoi IGS assay, were tested by 16S-23S IGS nPCR with the aim of sequencing products to identify other infecting Borrelia species.
Associations of infections and co-infections in ticks with province of origin, level of engorgement of the tick, host of origin, and tick instar were investigated by logistic regression in STATA version 11.0 for Windows (STATACorp, College Station, TX, USA). The most parsimonious multivariable model was created by backwards and forwards elimination and substitution of variables. Logistic regression models were used to investigate whether or not there were significant associations between infections of ticks with different pathogens. The level of significance throughout was P < 0.05.
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