Stomoxys flies harbor a low complexity microbiota dominated by environmental mastitis pathogens

Stomoxys (stable) flies have long been implicated as potential vectors of bovine mastitis-causing bacteria due to their close association with raw manure and soiled bedding in barn settings, which sustain large fly populations and serve as a reservoir for opportunistic bacteria. Despite this, the overall composition and diversity of bacterial communities associated with Stomoxys flies, especially in relation to their associated habitats, remain to be investigated. Here, we present the first culture-independent examination of Stomoxys­-associated bacterial communities through longitudinal sampling of fly and manure samples collected from two connected dairy facilities in South Central Wisconsin. Samples of Stomoxys flies and manure were collected on a weekly basis from July-September 2021. High-throughput 16S rRNA gene amplicon sequencing was then used to characterize and compare bacterial communities present on or within flies and in manure collected on the same sampling date. Bacterial alpha diversity was overall higher in manure samples as compared to fly samples, with manure-associated bacterial communities being dominated by members of the Bacteroidales and Eubacteriales. In contrast, flies harbored relatively low-complexity communities dominated by members of the Enterobacterales, Bacillales, and Lactobacillales. Many of the same bacterial strains were detected in both flies and manure collected from each facility, including clinically-relevant Escherichia, Enterococcus, and Staphylococcus spp. Clinically-relevant taxa were also not only present in flies but exhibited dramatically elevated abundances in fly samples as compared to manure samples. This research lays the foundation toward an improved understanding of the transmission of mastitis-causing bacteria in dairy barn settings.