A Cellular Model of Infection with Brucella melitensis in Ovine Macrophages: Novel Insights for Intracellular Bacterial Detection

Abstract

Intracellular bacteria provoking zoonoses, such as those of the genus Brucella, present a host cell tropism mostly limited to the monocyte/macrophage lineage, leading to chronic inflammatory reactions, difficult-to-eradicate-infections, and widespread prevalence among ruminants. Eradication of brucellosis has been based on programs that translate into a substantial financial burden for both the authorities and stockbreeders, if not strictly followed. To this end, we sought to create an in vitro cell model that could be utilized as future reference for adequately measuring the number of engulfed brucellae/cell, using peripheral blood-derived sheep macrophages infected with B. melitensis at decimal multiplicities of infection (MOI = 5000-5), to simulate the host cell/microorganism interaction and monitor bacterial loads up to 6 days post-infection. We show that the MOI = 5000 leads to high numbers of engulfed bacteria without affecting macrophages’ viability and that the minimum detection limit of our Real-Time PCR assay was 3.97 ± 5.58 brucellae/cell. Moreover, we observed a time-associated, significant gradual reduction in bacterial loads from Day 2 to Day 6 post-infection (p = 0.0013), as part of the natural bactericidal properties of macrophages. Overall, the work presented here constitutes a reliable in vitro cell model of Brucella melitensis for research purposes that can be utilized to adequately measure the number of engulfed brucellae/cell and provides insights towards future utilization of molecular biology-based methods for detection of Brucella.