Blocking primer improves detection of tick-borne pathogens in Ixodes scapularis (black-legged ticks) from a Lyme disease hotspot region in eastern Ontario, Canada.

Tick-borne pathogen (TBP) surveillance strategies that rely exclusively on targeted methods like PCR (PCR) or immunoblots do not benefit from strain-level sequence variation. Bacterial 16S rRNA metabarcoding offers more agnostic detection but is constrained in I. scapularis by the dominance of a maternally inherited endosymbiont, Rickettsia buchneri. Here we report the design and evaluation of three R. buchneri-specific blocking primers to suppress endosymbiont amplification during full-length 16S rRNA library preparation. Of these, primer 18F-Rb-C3 reduced R. buchneri relative abundance approximately 32-fold. We applied 18F-Rb-C3 with V4-16S metabarcode sequencing on 67 ticks collected from farm animals in Eastern Ontario and compared Borrelia species detection against qPCR. The V4-16S rRNA metabarcoding identified Borrelia species in 21 samples, whereas qPCR detected Borrelia in 24 samples and 11 samples were detected by both methods. Additionally, metabarcoding detected Anaplasma phagocytophilum in 12 samples, including seven samples coinfected with Borrelia, in the same assay. Variation relevant to strain surveillance was also detected by sequencing, though V4-16S was not sufficient to resolve closely related Borrelia genospecies or A. phagocytophilum variants. These findings demonstrate that blocking primer 18F-Rb-C3 enhances sensitivity of amplicon sequencing to the level of qPCR while also detecting other pathogens and sequence variants in a single assay.