Coinfection Ecology and Pathogen Emergence in a <i>Borrelia</i> -Endemic Landscape: Five Years of <i>Borrelia burgdorferi, Anaplasma phagocytophilum</i> , and <i>Babesia microti</i> Surveillance in Maryland

The emergence of tick-borne pathogens depends on ecological opportunity and barriers to persistence within vectors and hosts. <i>Borrelia burgdorferi</i> is firmly entrenched in the mid-Atlantic, whereas <i>Babesia microti</i> and <i>Anaplasma phagocytophilum</i> remain patchily distributed. Five years of integrated surveillance (2020-2024) at three Maryland sites allowed us to track <i>B. microti</i> and <i>A. phagocytophilum</i> establishment by screening questing <i>Ixodes scapularis</i> nymphs, <i>Peromyscus</i>-fed nymphs, and <i>Peromyscus leucopus</i> by qPCR, then contextualizing results with paired county-level human case data. <i>B. burgdorferi</i> was consistently detected in all sites and sample types, with prevalence stable at approximately 5-20% in questing nymphs and exceeding 30% in hosts, confirming long-term enzootic maintenance. By contrast, <i>B. microti</i> and <i>A. phagocytophilum</i> were initially sporadic but increased in prevalence, particularly in rodents and <i>Peromyscus</i>-fed ticks. Over time <i>A. phagocytophilum</i> prevalence significantly increased to above 20% in some <i>Peromyscus</i>-fed nymphal collections despite much lower prevalence in questing ticks, highlighting the early-warning value of bloodmeal-associated surveillance. Coinfections were rare, though enrichment of <i>B. burgdorferi</i> + <i>A. phagocytophilum</i> in <i>Peromyscus</i>-fed ticks suggests possible facilitation during early establishment. These results indicate that <i>B. microti</i> and <i>A. phagocytophilum</i> are actively emerging in Maryland, following their entrenchment in the Northeast and Upper Midwest. Combining surveillance from questing nymphal ticks, <i>Peromyscus</i>-fed nymphal ticks, and <i>P. leucopus</i> reservoir hosts provides a framework for detecting enzootic cycles before they appear in questing populations or human case counts, offering critical early-warning capacity for public health preparedness.