**6. Conclusions**

Thanks to its stealthy nature, upon infection, *Brucella* after entering inside a cell, reaches its intracellular niche, the ER, to replicate sheltered from detection by the immune system. This process is central to *Brucella* as it gives the bacterium the ability to maintain replicating-surviving cycles for long periods of time, even at low bacterial numbers, in its cellular niches. Eventually, *Brucella* will take advantage of the environment provided by its anatomic reservoirs, where the cellular niches reside, to disseminate to other organs, where high replication rates can occur. Of course, an organ reservoir would not exist without a pre-existing intracellular niche. It is also generally well interconnected with the other organs of the host to facilitate dissemination. Secretions and products of natural hosts of *Brucella*, livestock, and wildlife, contribute to contamination spreading. Figure 1 illustrates the journey of *Brucella* inside its hosts and recapitulates the intracellular replicative niche, cellular niches, organ reservoirs, and various hosts described in this report. In this challenging time where the world has seen the rapid emergence of a new viral zoonosis transmitted most probably from a pangolin, it is essential to better understand how another zoonosis, such as brucellosis, develops in its numerous wildlife hosts including bats, and livestock ones. Unraveling the molecular and cellular bases of *Brucella* host preference and reservoirs should be continued to preclude opportunities for *Brucella* to jump hosts.

Moreover, the persistence of viable furtive bacteria for extended periods of time highlights the ability of *Brucella* to maintain a chronic state, a feature that complicates brucellosis treatment, control, and eradication programs. A deeper understanding of the different organ reservoirs of *Brucella* should help to design new therapies, which would overcome the inability of current treatments to reach this surreptitious bacterium in certain cells and organs, as is the case for the bone marrow of infected patients.

*Brucella* niche is distributed in different anatomic reservoirs in the host and especially in some organs, such as the adipose tissue, the role of which in brucellosis is still speculative. This opens up new avenues of research that will undoubtedly contribute to a deeper knowledge of brucellosis and more generally of the mechanisms leading to the chronicity of intracellular pathogens.

**Figure 1.** Summary of *Brucella*'s cellular niches and reservoirs. The endoplasmic reticulum is the preferred intracellular niche for *Brucella*, but in some extravillous HLA-G+ trophoblasts, *B. abortus* and *B. suis* are located in lysosomal membrane-associated protein 1 (LAMP1)- and CD63-positive acidic inclusions. *Brucella* replicates in macrophages, dendritic cells, monocytes, trophoblasts, bone cells (osteoclasts, osteoblasts), granulocyte progenitors, adipocytes, and infects other cells such as neutrophils, lymphocytes, and erythrocytes. Some infected cells like neutrophils mediate *Brucella*´s immune response modulation and/or serve as a Trojan horse to disseminate and infect new organs. Several anatomical compartments are populated by *Brucella* infected cells. Organs with high replication rates (placenta, epididymis, mammary glands, lymph nodes, spleen, liver, lungs, and bone marrow) correlate with clinical manifestations of the disease. Once an adaptive immune response is achieved or granulomas contain the infection, *Brucella* develops chronicity and persists at low replication rates. An organ reservoir would not exist without a pre-existing intracellular niche. The structures and physiological characteristics of organ reservoirs allow *Brucella* to start new infection cycles within natural or accidental hosts. Although *Brucella* detection in wild sheep, goats, frogs, fox, bats, and rodents seems almost inconsequential, bacterial loads might be maintained within the host. The zoonotic potential of wildlife reservoirs is still unknown but represents an important risk of transmission to livestock or humans (Created with BioRender.com (accessed on 15 December 2020)).

**Author Contributions:** Conceptualization, G.G.-E., V.A.-G., S.M. and J.-P.G.; supervision, S.M. and J.-P.G.; writing-original draft preparation, G.G.-E. and S.M.; project administration, S.M. and J.-P.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** Work is supported by institutional funding from the Centre National de la Recherche Scientifique (CNRS) and the Institut National de la Santé et de la Recherche Médicale (INSERM), and by the Fondation pour la Recherche Médicale (FRM) gran<sup>t</sup> DEQ20170336745, "Laboratories of excellence" programme (Labex INFORM, ANR-11-LABX-0054), the Excellence Initiative of Aix-Marseille Université (AMU) (A\*MIDEX), a French "Investissements d'Avenir" programme. GGE is a recipient of o fellowship from the Fondation de Coopération Scientifique "Méditerranée Infection" and from Campus France.

**Acknowledgments:** Authors thank all members of the JPG lab for their support.

**Conflicts of Interest:** The authors declare no conflict of interest.
