Synanthropic flies harbor a distinct, pathogen-enriched microbial compartment in livestock landscapes

Zoonotic and other livestock-associated pathogens move readily within animal production systems, yet their dispersal into surrounding environments is poorly characterized. We evaluated whether synanthropic filth flies act as spatially integrating sentinels of livestock-associated pathobiomes. From 30 roadside sites in six Texas counties stratified by livestock production intensity, we collected 3,373 Calliphoridae spp. and 12 Musca domestica, together with co-located soil, water, plant, and fecal samples, and characterized 195 metagenomes (122 insect, 73 environmental). Using diversity, differential-abundance, machine-learning, indicator-species, and network analyses, we found three robust patterns. First, fly metagenomes form a microbial compartment compositionally distinct from co-located environmental matrices (PERMANOVA R{superscript 2} = 0.094, F = 19.81, P = 0.001) and carry markedly higher relative abundances of pathogen-associated taxa, so insect sampling is complementary to, not redundant with, environmental sampling. Second, the dominant landscape signal is categorical rather than graded: cattle presence, not a Low-Medium-High density gradient, most strongly correlated with community structure, with the ruminant-associated anaerobe Peptostreptococcus russellii as the most reproducible biomarker. Third, intensification produced taxon-level enrichments (notably the feedlot pathogen Trueperella pyogenes) and progressive co-occurrence-network fragmentation, without a monotonic dose-response. A curated 12-pathogen panel failed to classify landscape context, whereas whole-community analysis succeeded. Filth flies thus provide a pragmatic, spatially integrated readout that distinguishes low-impact from livestock-impacted landscapes, offering a defensible tool for One Health pathogen surveillance. IMPORTANCEZoonotic and other livestock-associated pathogens emerging from animal production are among the most pressing threats to global health but tracking how they spill into the wider environment is difficult and expensive. We show that common filth flies, abundant, easy to trap, and free ranging across farms, fields, and human spaces, act as living samplers that concentrate and report the microbial signature of nearby livestock. Sampling flies captured information that soil and water sampling missed, and a single binary signal, the presence or absence of cattle, was associated with the microbial community more strongly than any measure of animal density. Narrow tests targeting a short curated list of pathogens were not successful in differentiating sites, while analyzing the whole microbial community provided more discriminating power. These findings support a practical, low-cost strategy in which insects serve as biological sentinels for the detection of livestock-associated pathogens across agricultural landscapes.