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

The emergence of tick-borne pathogens depends on ecological opportunity and barriers to persistence within vectors and hosts. Borrelia burgdorferi is firmly entrenched in the mid-Atlantic, whereas Babesia microti and Anaplasma phagocytophilum remain patchily distributed. Five years of integrated surveillance (2020-2024) at three Maryland sites allowed us to track B. microti and A. phagocytophilum establishment by screening questing Ixodes scapularis nymphs, Peromyscus-fed nymphs, and Peromyscus leucopus by qPCR, then contextualizing results with paired county-level human case data. B. burgdorferi 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, B. microti and A. phagocytophilum were initially sporadic but increased in prevalence, particularly in rodents and Peromyscus-fed ticks. Over time A. phagocytophilum prevalence significantly increased to above 20% in some Peromyscus-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 B. burgdorferi + A. phagocytophilum in Peromyscus-fed ticks suggests possible facilitation during early establishment. These results indicate that B. microti and A. phagocytophilum are actively emerging in Maryland, following their entrenchment in the Northeast and Upper Midwest. Combining surveillance from questing nymphal ticks, Peromyscus-fed nymphal ticks, and P. leucopus 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. IMPORTANCEUnderstanding why some tick-borne pathogens become ecosystem entrenched while others remain sporadic is central to predicting human disease emergence. By combining surveillance of questing nymphal Ixodes ticks, Peromyscus-fed nymphal ticks, and Peromyscus leucopus reservoir hosts across five years in Maryland, we show that Babesia microti and Anaplasma phagocytophilum remain in the early stages of ecological entrenchment whereas Borrelia burgdorferi is deeply established. This integrated approach demonstrates how pathogen biology within the tick shapes field prevalence and highlights Peromyscus-fed ticks as a powerful xenodiagnostic early-warning tool for detecting emerging pathogens before they are reflected in questing populations or human case data.