Phylogeography and Genetic Diversity of Francisella tularensis subsp. holarctica in France (1947-2018)

In France, tularemia is caused by Francisella tularensis subsp. holarctica and is a sporadic disease affecting mainly wildlife animals and humans. F. tularensis species presents low genetic diversity that remains poorly described in France, as only a few genomes of isolates from the country are available so far. The objective of this study was to characterize the genetic diversity of F. tularensis in France and describe the phylogenetic distribution of isolates through whole-genome sequencing and molecular typing. Whole genomes of 350 strains of human or animal origin, collected from 1947 to 2018 in France and neighboring countries, were sequenced. A preliminary classification using the established canonical single nucleotide polymorphism (canSNP) nomenclature was performed. All isolates from France (except four) belonged to clade B.44, previously described in Western Europe. To increase the resolution power, a whole-genome SNP analysis was carried out. We were able to accurately reconstruct the population structure according to the global phylogenetic framework, and highlight numerous novel subclades. Whole-genome SNP analysis identified 87 new canSNPs specific to these subclades, among which 82 belonged to clade B.44. Identifying genomic features that are specific to sublineages is highly relevant in epidemiology and public health. We highlighted a large number of clusters among a single clade (B.44), which shows for the first time some genetic diversity among F. tularensis isolates from France, and the star phylogeny observed in clade B.44-subclades revealed that F. tularensis biodiversity in the country is relatively recent and resulted from clonal expansion of a single population. No association between clades and hosts or clinical forms of the disease was detected, but spatiotemporal clusters were identified for the first time in France. This is consistent with the hypothesis of persistence of F. tularensis strains found in Western Europe in the environment, associated with slow replication rates. Moreover, the presence of identical genotypes across long periods of time, and across long distances, supports this hypothesis but also suggests long-distance dispersal of the bacterium.This work was supported by the French National Research Agency (ANR) and the Direction Générale de l’Armement (DGA) (No. ANR-15-ASTR-0021-01). MK is a Ph.D. student co-supported by Université Paris-Est and DGA grants

High-resolution melting PCR assay as a powerful tool for the epidemiological surveillance of tularemia in Western Europe

International audienc

Human Brucellosis in Maghreb: Existence of a Lineage Related to Socio-Historical Connections with Europe

<div><p>Despite control/eradication programs, brucellosis, major worldwide zoonosis due to the <i>Brucella</i> genus, is endemic in Northern Africa and remains a major public health problem in the Maghreb region (Algeria/Morocco/Tunisia). <i>Brucella melitensis</i> biovar 3 is mostly involved in human infections and infects mainly small ruminants. Human and animal brucellosis occurrence in the Maghreb seems still underestimated and its epidemiological situation remains hazy. This study summarizes official data, regarding <i>Brucella melitensis</i> infections in Algeria, from 1989 to 2012, with the purpose to provide appropriate insights concerning the epidemiological situation of human and small ruminant brucellosis in Maghreb. Algeria and Europe are closely linked for historical and economical reasons. These historical connections raise the question of their possible impact on the genetic variability of <i>Brucella</i> strains circulating in the Maghreb. Other purpose of this study was to assess the genetic diversity among Maghreb <i>B. melitensis</i> biovar 3 strains, and to investigate their possible epidemiological relationship with European strains, especially with French strains. A total of 90 <i>B. melitensis</i> biovar 3 Maghreb strains isolated over a 25 year-period (1989–2014), mainly from humans, were analysed by MLVA-16. The obtained results were compared with genotypes of European <i>B. melitensis</i> biovar 3 strains. Molecular assays showed that Algerian strains were mainly distributed into two distinct clusters, one Algerian cluster related to European sub-cluster. These results led to suggest the existence of a lineage resulting from socio-historical connections between Algeria and Europe that might have evolved distinctly from the Maghreb autochthonous group. This study provides insights regarding the epidemiological situation of human brucellosis in the Maghreb and is the first molecular investigation regarding <i>B. melitensis</i> biovar 3 strains circulating in the Maghreb.</p></div

<i>B. melitensis</i> bv 3 HGDI values for each MLVA-16 genetic marker (from this study and from published data).

<p>Hunter Gaston Diversity Index (HGDI): Measure of the variation of the number of repeats at each locus. Ranges from 0.0 (no diversity) to 1.0 (complete diversity); Confidence Interval: Precision of the Diversity Index, expressed as 95% upper and lower boundaries; K: Number of different repeats present at this locus in this sample set; max(pi): Fraction of samples that have the most frequent repeat number in this locus (range 0.0 to 1.0); n: strains number according to the considered locus; in bold: locus more variable. *: <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115319#pone.0115319-AlDahouk1" target="_blank">[17]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115319#pone.0115319-LeFleche1" target="_blank">[25]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115319#pone.0115319-Mick1" target="_blank">[31]</a>.</p><p><i>B. melitensis</i> bv 3 HGDI values for each MLVA-16 genetic marker (from this study and from published data).</p

MST of clustered MLVA-11 genotypes of the worldwide <i>B. melitensis</i> bv 3 isolates.

<p>The MST was constructed with a categorical coefficient (with panel weights). Size of circles reflects the number of isolates with a particular MLVA genotype. Width of the line reflects the genetic distance between the genotypes (heavy short lines connect SLVs, thin longer lines connect DLVs). Each country/group is assigned a different colour, <i>i.e.</i> isolates from Algeria are coloured in red, isolates from Morocco in green, isolates from Tunisia in yellow, isolates from <i>largo sensu</i> Maghreb in light blue, West Mediterranean isolates from Europe in blue, East Mediterranean isolates (n = 267) in khaki, American isolates in orange and the <i>B. melitensis</i> bv 1 reference strain 16 M in grey. Regarding the West Mediterranean group, the MLVA-11 genotypes identified in this study were indicated (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115319#pone.0115319.s002" target="_blank">S1 Table</a>).</p

Concatenated dendrogram of clustered MLVA-16 genotypes of Europe and Maghreb isolates.

<p>The dendrogram was constructed with a categorical coefficient and UPGMA algorithm. Clusters are coded as A and B, and sub-clusters as A1–A2. MLVA-16 genotypes of all investigated strains and strain details are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115319#pone.0115319.s001" target="_blank">S1 Figure</a>.</p

Incidence of <i>B. melitensis</i> human and animal brucellosis in Algeria, <i>vs</i> incidence of human brucellosis in France and number of human cases imported from Algeria to France.

<p>A: Incidence of human and animal brucellosis in Algeria (1/100,000 inhabitants); B: Incidence of human brucellosis in France (1/100,000 inhabitants) and number of human cases imported from Algeria to France The x-axis indicates the year. The y-axis represents (A) incidence of human brucellosis or number of new animal cases reported in Algeria and (B) incidence of human brucellosis in France or number of new human <i>B. melitensis</i> cases imported from Algeria to France.</p

Phylogeography and epidemiology of Brucella suis biovar 2 in wildlife and domestic swine

Swine brucellosis due to Brucella suis biovar 2 (bv2) is enzootic in wild boar and hare in continental Europe and may cause major economic losses to the pig industry, mainly in free-ranged pig farms. The high nucleotide identity found among the B. suis biovar 2 isolates has long hindered the full understanding of the epidemiology and the phylogeography of the disease. Here, we used multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) and whole-genome analysis to identify single-nucleotide polymorphisms (SNPs) in order to gain insights from the largest B. suis bv2 dataset analyzed so far composed of domestic pigs and wildlife isolates collected throughout Europe since the 1970s. We found four major clades with a specific phylogeographic pattern. The Iberian clade contains isolates exclusively from the Iberian Peninsula. The Central European clade includes most isolates from France, Northern Italy, Switzerland and an important proportion of those of Northern Spain. The Eastern European clade clustered isolates from Croatia and Hungary mainly but also from areas of France, Germany, Italy and Poland. Finally, a separated Sardinian clade grouped three isolates from this island. At fine scale, MLVA demonstrated an endemic status of the infection in Europe and it allowed tracking a large outbreak formed by different farms from Spain linked to the same infection source. The whole genome SNP analysis showed that the strains form genetically distinct clades, shared between wild boar and pigs, in agreement with the MLVA clades. Interestingly, all hare isolates clustered together within two groups composed exclusively of wildlife isolates. Our results support the hypothesis that maintenance and spread of B. suis bv2 in Europe is a dynamic process linked to the natural expansion of wild boar as the main wild reservoir of the infection, while spread over long distances is found largely dependent on anthropogenic activities.Unpublishe