Phylogenetic analysis of Mycobacterium bovis reveals animal and zoonotic tuberculosis spread between Morocco and European countries

Abstract

Livestock production is a fundamental pillar of the Moroccan economy. Infectious diseases of cattle and other species represent a significant threat to the livestock industry, animal health, and food safety. Bovine tuberculosis (bTB), mainly caused by Mycobacterium bovis, generates considerable direct and indirect economic losses, and an underestimated human health burden caused by zoonotic transmission. Previous studies have suggested likely M. bovis transmission links between Morocco and Southern Europe, however, limitations inherent with the methods used prevented definitive conclusions. In this study, we employed whole genome sequencing analysis to determine the genetic diversity of the first 55 M. bovis whole-genomes in Morocco and to better define the phylogenetic links between strains from Morocco and a large dataset from related and neighboring countries. With a total of 780 M. bovis sequences extracted from cattle, wildlife or humans and representing 36 countries, we discovered two new M bovis spoligotypes in Morocco and that the Moroccan clonal complexes are classified as belonging to Europe or Unknown, supporting previous studies that the Sahara Desert might be playing a key role in preventing M. bovis transmission between North Africa and Sub-Saharan Africa. Furthermore, our analysis showed a close M. bovis genetic relationship between cattle from Morocco and cattle from Spain, France, Portugal and Germany, and from cattle in Morocco and humans in Italy, Germany, and the UK. These results suggest that animal trade and human migration between Morocco and these countries might be playing a role in disease transmission. Our study benefits from a large sample size and a rich dataset that includes sequences from cattle, wildlife and humans from Morocco and neighboring countries, enabling the delineation of M. bovis genetic links across countries and host-species. Our study calls for further investigation of animal and zoonotic TB spread in Morocco and in other countries, which is important to inform future TB control measures at the animal-human interface.

Fig 1. Map showing the slaughterhouse locations from where tissue samples with evidence of bTB-like lesions were collected from cattle. The brown outline shows the Sahara Desert. We used the rnaturalearth package in R to download world country polygons from Natural Earth, sub-setting to Northern Africa. We produced the map using the sf and ggplot packages in R, plotting Oujda, Rabat, and Casablanca points using latitude and longitude data [–48]. The data to build the map in Fig 1 came from here: https://www.rdocumentation.org/packages/rnaturalearth/versions/1.0.1/topics/countries.

Fig 2. Distribution of Mycobacterium bovis isolates from North Africa and Sub-Saharan Africa based on clonal complexes/lineages.

Fig 3. Maximum-likelihood phylogenetic tree with all Mycobacterium bovis isolates used in this study.

The phylogenetic tree was generated using the GTR-CAT substitution model and presents all the analyzed M. bovis isolates used in the study (n = 725), with information about countries (inner circle), clonal complex (middle circle) and lineages (outer circle) color coded. Moroccan isolates are indicated with bold marked tree branches, and human isolates are indicated with a red dot. The areas filled with a light grey shade indicate the groups that include Moroccan isolates.

Fig 4. High-resolution maximum-likelihood phylogenetic tree of groups 1–8.

The legend indicates country of origin based on the highlighted isolate color. The scale bar represents the branch length in SNPs. Within this figure, eight panels illustrate the genetic relationships among M. bovis isolates. Fig 4A highlights the genetic connection between Moroccan and Spanish M. bovis isolates from cattle, alongside one of the seven Canadian M. bovis isolates analyzed. Fig 4B reveals genetic relationships between M. bovis isolates from Moroccan cattle and UK human in the top red box, while the bottom red box showcases the close genetic proximity of M. bovis isolates between Moroccan and Spanish cattle. Fig 4C emphasizes genetic proximity between M. bovis isolates from Moroccan and Spanish cattle in the top red box, with the second box displaying clustering of M. bovis isolates between Moroccan and Portuguese cattle. The third and fourth red boxes in Panel 3 show that M. bovis from Moroccan cattle are genetically related with M. bovis isolates collected from humans in Germany (including one that shares their Most Recent Common Ancestor with 20 SNPs apart). The bottom box also highlights another closely related relationship between a M. bovis isolate from Moroccan cattle a M. bovis isolate from human in Germany, as well as between M. bovis in Moroccan cattle and M. bovis in Algerian cattle. Fig 4D demonstrates the close genetic relationship between M. bovis cattle isolates from Morocco, Spain, and Portugal. Fig 4E displays genetic relationships between M. bovis isolates from Moroccan cattle and Spanish cattle and Moroccan cattle and humans in Germany. Fig 4F does not show close genetic links between M. bovis isolates from Morrocan cattle and cattle or humans from other countries. Fig 4G shows a close genetic relationship between M. bovis from Moroccan cattle and three humans from Germany. Fig 4H’s red box illustrates the genetic clustering of M. bovis isolates between cattle from Morocco and humans from Italy and Switzerland. The bottom red box highlights the close genetic relationship of M. bovis isolates between cattle in Morocco and a human in Italy, as well as the close relationship of these isolates with one from cattle in Algeria.