16S profiling of the vaginal microbiome in Brucella-vaccinated pregnant ewes

Live attenuated vaccines based on Brucella melitensis 16M?wzm and Rev1?wzm mutants are promising candidates for controlling brucellosis in small ruminants. Whereas Rev1?wzm has demostrated complete safety in pregnant ewes, 16M?wzm remains a concern, as mid-gestation inoculation triggered reactivation in half of the periparturient ewes. This study aimed to characterize the vaginal microbiome dynamics associated with each B. melitensis ?wzm mutant during pregnancy and to evaluate the predictive value of microbiome profiling for pathogen detection at peripartum. Serial vaginal swabs from a cohort of ewes inoculated at mid-pregnancy with Rev1?wzm, 16M?wzm or PBS were collected at key stages of B. melitensis placental infection (5, 9, and 11–12 weeks post-inoculation, PI). Bacterial community composition was assessed by high-throughput sequencing of the 16S rRNA V3–V4 region. In agreement with culture results, neither vaccine strain was detected in the vaginal microbiota during pregnancy. Importantly, the vaginal microbiome of Rev1?wzm-inoculated ewes remained stable at peripartum and no Brucella DNA was detected in these samples. In contrast, Brucella DNA was consistently detected in the vaginal microbiome of all ewes experiencing 16M?wzm reactivation at delivery (11–12 weeks PI), but not in the ewe that remained negative. These findings confirm the superior safety of Rev1?wzm over 16M?wzm and highlight the potential of microbiome monitoring as a rapid, non-invasive tool for Brucella detection under field conditions. Furthermore, microbiome sequencing emerges as a valuable approach for studying Brucella infections in natural hosts, with potential applications for controlling this major zoonosis within the One Health framework.