Abstract
Because of the importance for public health and damages induced by Brucella (B.) infection in livestock farming, scientists and field workers try to control and eradicate the disease in animals. However, existing vaccines do not meet all the safety criteria for animals. Currently, the only vaccines able to induce a protection to natural domestic animal hosts against brucellosis are live attenuated vaccines (LAVs), as B.abortus S19 and B. melitensis Rev.1. Unfortunately, they have some risks of human contamination, interfere with diagnostic tests and importantly, they can induce abortions in animals. Developing safer vaccines for animals would help to reduce the impact of brucellosis. First generation LAVs relied on empirical and somewhat unpredictable attenuation. Recent advances in genetics make it possible to envisage the rational construction of LAVs. Our objective is to achieve by Transposon Sequencing (Tn-seq) a functional map of the Brucella melitensis genome in order to construct a candidate LAV capable of persisting long enough to induce protective immunity but incapable of persisting in tissues or invading the placenta in pregnant animals. For this purpose, Tn-seq analyses were performed under different conditions in the mouse model. To generate the Tn-seq, we have generated a library of about 106 transpositional mutants. We have identified in mice genes required for the persistence of B. melitensis in the lung after an intranasal inoculation and in the spleen after an intraperitoneal injection. By using these protocols, we avoided the bottleneck which is a limit of the method. Our results show that essential genes for the persistence ofBrucella vary according to the analyzed tissues. Many of these genes are involved in bacterial metabolism, suggesting that Brucella has different nutritional requirements depending on the colonized tissues. Tn-seq predictions were validated by constructing markerless deletion mutants and testing them in mice. On this basis, several candidate vaccines have been selected and are in the validation phase in mice.
