Metagenomic surveillance of tick-borne pathogens and microbiomes in Huntingdon County, Pennsylvania.

The rise in tick populations across the United States has contributed to a surge in tick-borne diseases, with Pennsylvania ranking among the highest in reported cases. To better understand local pathogen prevalence and microbial community structure, an integrative study of ticks collected from ten recreational trails in Huntingdon County, Pennsylvania during the summer of 2023 was conducted. A total of 96 ticks were sampled, with 33 PCR-positive specimens selected for shotgun metagenomic sequencing. Pathogen screening via qPCR detected Borreliella burgdorferi, Borrelia miyamotoi, Babesia spp., and Anaplasma phagocytophilum. Shotgun metagenomics revealed a broader diversity of tick-borne pathogens, including Rickettsia and Ehrlichia spp., and demonstrated increased sensitivity by detecting low-abundance pathogens in samples that were PCR-negative. Co-infections were common, and multivariate statistical analysis identified significant associations between environmental variables (e.g., humidity, time of day, land cover) and microbial diversity and predicted gene function. Notably, diversity was higher in ticks collected during early afternoon and from northern sites. Co-occurrence network analysis showed Rickettsia as a central taxon with multiple significant positive associations with other microbes while other pathogens were largely absent or peripheral. These findings underscore the enhanced resolution of metagenomic approaches for pathogen detection and the value of combining molecular surveillance with ecological metadata. Our study provides critical insights into local tick microbiomes and pathogen prevalence, which may inform public health interventions and vector management strategies in central Pennsylvania.