Pathogenesis of Brucella abortus and Brucella melitensis in bovine and ovine-derived trophoblasts and macrophages and impaired intracellular trafficking of the Rev1 vaccine strain.

Brucella spp. are Gram-negative facultative intracellular pathogens responsible for brucellosis, a widespread zoonosis. Among them, B. melitensis and B. abortus are the most epidemiologically relevant species, primarily infecting sheep and goats, and cattle, respectively. Upon infection, brucellae multiply within phagocytes and develop placental tropism, particularly targeting trophoblasts. Most studies of Brucella pathogenesis and vaccine development have relied on murine or human-derived epithelial and macrophagic cell lines, which may not fully reproduce the natural ruminant host. Here, we investigated whether the intracellular behaviour described for Brucella in non-natural-host models also occurs in ruminant trophoblasts (AH-1 and F3 cells) and macrophages (BoMac cells). Using confocal immunofluorescence microscopy and bacteriological assays, we observed similar infection rates for B. abortus and B. melitensis. By 24 hours post-infection (hpi), both species had transitioned to LAMP1-negative Brucella-containing vacuoles, with active replication occurring within calnexin-positive compartments. These results confirm that the Brucella intracellular trafficking previously described in non-natural-host models is conserved in these ruminant cells. Additionally, we characterised for the first time the intracellular behaviour of the Rev1 vaccine strain. Although its initial rate of infection was comparable to B. melitensis 16M, Rev1 exhibited a delay in LAMP1-exclusion from the Brucella-containing vacuole, with most bacteria remaining in LAMP1-positive compartments at 24 hpi, and showed delayed initiation of replication. These findings, together with slower in vitro growth, suggest that the Rev1 attenuation results from both delayed intracellular trafficking and impaired intracellular fitness. Our results advance the understanding of Brucella pathogenesis in natural-host and shed light on Rev1 attenuation, offering key insights to enhance current brucellosis vaccines.